Hydrazide-containing cftr inhibitor compounds and uses thereof

ABSTRACT

The invention provides compositions, pharmaceutical preparations and methods for inhibition of cystic fibrosis transmembrane conductance regulator protein (CFTR) that are useful for the study and treatment of CFTR-mediated diseases and conditions. The compositions and pharmaceutical preparations of the invention may comprise one or more hydrazide-containing compounds, and may additionally comprise one or more pharmaceutically acceptable carriers, excipients and/or adjuvants. The methods of the invention comprise, in certain embodiments, administering to a patient suffering from a CFTR-mediated disease or condition, an efficacious amount of a hydrazide-containing compound. In other embodiments the invention provides methods of inhibiting CFTR that comprise contacting cells in a subject with an effective amount of a hydrazide-containing compound. In addition, the invention features a non-human animal model of CFTR-mediated disease which model is produced by administration of a hydrazide-containing compound to a non-human animal in an amount sufficient to inhibit CFTR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/093,749, now allowed, which has a filing date of Mar. 29, 2005, whichclaims the benefit U.S. Provisional Application No. 60/557,930, filedMar. 30, 2004, all of which applications are incorporated herein byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant nos.HL73854, EB00415, EY13574, DK35124, DK43840, and UC1 A1062530-01 awardedby the National Institutes of Health. The government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

The cystic fibrosis transmembrane conductance regulator protein (CFTR)is a cAMP-activated chloride (Cl⁻) channel expressed in epithelial cellsin mammalian airways, intestine, pancreas and testis. CFTR is thechloride-channel responsible for cAMP-mediated Cl⁻ secretion. Hormones,such as a β-adrenergic agonist, or a toxin, such as cholera toxin, leadsto an increase in cAMP, activation of cAMP-dependent protein kinase, andphosphorylation of the CFTR Cl⁻ channel, which causes the channel toopen. An increase in cell Ca²⁺ can also activate different apicalmembrane channels. Phosphorylation by protein kinase C can either openor shut Cl⁻ channels in the apical membrane. CFTR is predominantlylocated in epithelia where it provides a pathway for the movement of Cl⁻ions across the apical membrane and a key point at which to regulate therate of transepithelial salt and water transport. CFTR chloride channelfunction is associated with a wide spectrum of disease, including cysticfibrosis (CF) and with some forms of male infertility, polycystic kidneydisease and secretory diarrhea.

The hereditary lethal disease cystic fibrosis (CF) is caused bymutations in CFTR. Observations in human cystic fibrosis (CF) patientsand CF mouse models indicate the functional importance of CFTR inintestinal and pancreatic fluid transport, as well as in male fertility(Grubb et al., 1999, Physiol. Rev. 79:S193-S214; Wong, P. Y., 1997, Mol.Hum. Reprod. 4:107-110). However, the mechanisms remain unclear by whichdefective CFTR produces airway disease, which is the principal cause ofmorbidity and mortality in CF (Pilewski et al., 1999, Physiol. Rev.79:S215-S255). Major difficulties in understanding airway disease in CFinclude the inadequacy of CF mouse models, which manifest little or noairway disease, the lack of large animal models of CF, and the limitedavailability of human CF airways that have not been damaged by chronicinfection and inflammation. High-affinity, CFTR-selective inhibitorshave not been available to study airway disease mechanisms in CF or tocreate the CF phenotype in large animal models.

High-affinity CFTR inhibitors also have clinical applications in thetherapy of secretory diarrheas and cystic kidney disease, and ininhibiting male fertility. Several CFTR inhibitors have been discovered,although most of which have a weak potency and lack CFTR specificity.The oral hypoglycemic agent glibenclamide inhibits CFTR Cl⁻ conductancefrom the intracellular side by an open channel blocking mechanism(Sheppard & Robinson, 1997 J. Physiol., 503:333-346; Zhou et al., 2002,J. Gen. Physiol., 120:647-662) at high micromolar concentrations whereit affects other Cl⁻ and cation channels (Edwards & Weston, 1993; Rabeet al., 1995, Br. J. Pharmacol., 110: 1280-1281; Schultz et al., 1999,Physiol. Rev., 79:S109-S144). Other non-selective anion transportinhibitors including diphenylamine-2-carboxylate (DPC),5-nitro-2(3-phenylpropyl-amino)benzoate (NPPB), and flufenamic acid alsoinhibit CFTR by occluding the pore at an intracellular site (Dawson etal., 1999, Physiol. Rev., 79:S47-S75; McCarty, 2000, J. Exp. Biol.,203:1947-1962).

There is accordingly a need for CFTR inhibitors, particularly those thatare water-soluble. The present invention addresses these needs, as wellas others, and overcomes deficiencies found in the background art.

Literature

Ma et al., 2002, J. Clin. Invest., 110:1651-1658 describes athiazolidinone class of CFTR inhibitor.

SUMMARY OF THE INVENTION

The invention provides compositions, pharmaceutical preparations andmethods for inhibition of cystic fibrosis transmembrane conductanceregulator protein (CFTR) that are useful for the study and treatment ofCFTR-mediated diseases and conditions. The compositions andpharmaceutical preparations of the invention may comprise one or morehydrazide-containing compounds, and may additionally comprise one ormore pharmaceutically acceptable carriers, excipients and/or adjuvants.The methods of the invention comprise, in certain embodiments,administering to a patient suffering from a CFTR-mediated disease orcondition, an efficacious amount of a hydrazide-containing compound. Inother embodiments the invention provides methods of inhibiting CFTR thatcomprise contacting cells in a subject with an effective amount of ahydrazide-containing compound. In addition, the invention features anon-human animal model of CFTR-mediated disease which model is producedby administration of a hydrazide-containing compound to a non-humananimal in an amount sufficient to inhibit CFTR.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the hydrazide-containing compounds as more fully describedbelow.

BRIEF DESCRIPTIONS OF THE DRAWINGS

The invention will be more fully understood by reference to thefollowing drawings, which are for illustrative purposes only.

FIG. 1A is a schematic representation of a screening technique used fordetection of CFTR inhibitors. CFTR was maximally stimulated by multipleagonists in stably transfected epithelial cells co-expressing human CFTRand a yellow fluorescent protein (YFP) having Cl⁻/I⁻ sensitivefluorescence. After addition of a test compound, I⁻ influx was inducedby adding an I⁻ containing solution.

FIG. 1B shows chemical structures of CFTR inhibitors identified by thescreening technique of FIG. 1A.

FIG. 1C is a graph representing relative fluorescence versus time usingthe screening technique of FIG. 1A for the CFTR inhibitorN-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide (referred to herein as GlyH-101) at several concentrations.

FIG. 1D is a graph representing GlyH-101 inhibition of short-circuitcurrent in permeabilized FRT cells expressing human CFTR. CFTR wasstimulated by 100 μM CPT-cAMP.

FIG. 2A is a graph representing the time course of inhibition showingCFTR-mediated I⁻ transport rates at different times after addition of 10μM GlyH-101.

FIG. 2B is a graph representing the time course of inhibition reversalshowing I⁻ transport rates at different times after washout of GlyH-101.

FIG. 2C is a graph representing iodide influx by GlyH-101 (50 μM) afterCFTR stimulation by indicated agonists (50 μM). Filled bars showagonist, and open bars show agonist with GlyH-101.

FIG. 3A provides chemical structures of a class of GlyH-101 analogs withsites of modification indicated with brackets.

FIG. 3B depicts the reaction scheme for the synthesis of GlyH-101,N-(6-quinolinyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide (referred to herein as GlyH-126),3,5-dibromo-2,4-di-hydroxy-[2-(2-napthalenamine)aceto]benzoic acidhydrazide (referred to herein as GlyH-201), andN-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methyl]glycinehydrazide (referred to herein as GlyH-301). Reagents and conditions: (a)ICH₂COOEt, NaOAc, 95° C.; (b) N₂H₄.H₂O EtOH/reflux; (c)3,5-di-Br-2,4-di-OH-Ph-CHO, EtOH/reflux; (d)3,5-di-Br-2,4-di-OH-Ph-COCl, pyridine, 22° C.; (e) N₂H₄.H₂O, Pd/C (10%),DMF/reflux; (0 glyoxalic acid, 10° C.; (g) Na₂BH₃CN/CH₃CN, 48 h; dryHCl, EtOH.

FIG. 3C is a graph representingN-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]oxamic acidhydrazide (referred to herein as OxaH-110) inhibition of short-circuitcurrent permeabilized FRT cells expressing human CFTR (right panel) andthe structure of OxaH-110 (left panel). CFTR was stimulated by 100 μMCPT-cAMP.

FIG. 4A is a graph illustrating of GlyH-101 inhibition measured inwhole-cell patch clamp experiments on FRT cells expressing human CFTR.Whole-cell membrane currents were evoked by voltages from −100 to +100mV in 20 mV steps after maximal CFTR stimulation by 5 μM forskolin. Thegraph on the left represents measurements before GlyH-101 was added andthe graph on the right represents measurements after GlyH-101 was added.

FIG. 4B is a graph representing current-voltage relationships in theabsence of inhibitors (control, open circles), after addition of 10 μM(filled squares) and 30 μM GlyH-101 (filled circles), after washout of10 μM GlyH-101 (recovery, triangles) and after addition of 5 μMCFTR_(inh)-172 (filled circles).

FIG. 4C is a graph illustrating of dose-response relationshipsdetermined for GlyH-101 at the indicated membrane potentials.

FIG. 4D is a graph illustrating of representative cell-attachedpatch-clamp recordings showing CFTR single channel activity at GlyH-101concentrations of 0, 0.4 and 5 μM. Dashed lines show zero current level(channels closed) with downward deflections indicating channel openings(Cl⁻ ions moving from pipette into the cell). Pipette potential was −60mV.

FIG. 5A is a graph illustrating the pH-dependent absorbance changes(right panel) of the chemical compounds (10 μM) (corresponding chemicalstructures, left panel) in NaCl (100 mM) containing MES, HEPES, boricacid, and citric acid (each 10 mM) titrated to different pH usingHCl/NaOH. Absorbance changes measured at analytical wavelengths of 346,348, 346, and 236 nm (top to bottom).

FIG. 5B is a representation of deduced ionic equilibria of GlyH-101showing pKa values.

FIG. 6A is a graph illustrating GlyH-101 inhibition in a nasal potentialdifference (PD) recording showing responses to amiloride and low Cl⁻solutions (left panel) or averaged PD values (right panel, mean±SE,n=5). Where indicated the low Cl⁻ solutions contained forskolin withoutor with GlyH-101.

FIG. 6B is a graph representing a paired analysis of experiments as inFIG. 6A showing PD changes (ΔPD) for the forskolin effect, forskolin andCFTR_(inh)-172, and forskolin and GlyH-101.

FIG. 6C is a graph illustrating of a change in PD (mean±SE) in a seriesof low Cl⁻ induced hyperpolarization experiments (left panel) orforskolin induced hyperpolarization (right panel) in which solutionscontained either 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)or GlyH-101 (* P<0.005 for reduced ΔPD compared to control).

FIG. 7A is a graph illustrating GlyH-101 inhibition of short-circuitcurrent after CFTR stimulation in T84 cells (top panel), human airwaycells (middle panel), and isolated mouse ileum (bottom panel). Followingconstant baseline current, amiloride (10 μM, apical solution) andCPT-cAMP (0.1 mM, both solutions) were added, followed by indicatedconcentrations of GlyH-101 (both solutions).

FIG. 7B is a graph representing GlyH-101 inhibition of fluid secretionin a closed intestinal loop model of cholera toxin-induced fluidsecretion. Intestinal lumenal fluid, shown as loop weight/length (gm/cm,SE, 6 mice), measured at 4 hours after injection of saline (control),cholera toxin (1 μg) or cholera toxin+GlyH-101 (0.25 μg).

FIG. 8 provides chemical structures of a class of non-absorbable molonicacid dihydrazide (denoted as MalH-x) analogs of glycine hydrazidecompounds of the invention.

FIG. 9 depicts the reaction scheme for the synthesis of the polarnon-absorbable CFTR inhibitors2-naphthalenylamino-bis[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propanedioicacid dihydrazide (MalH-1),2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][(2,4-disodium-disulfophenyl)methylene]propanedioicacid dihydrazide (MalH-2), and2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-(4-sodium-sulfophenyl)-thioureido]propanedioicacid dihydrazide (MalH-3). Reagents and conditions: (a) diethylbromomalonate, NaOAc, 90° C., 8 hours, 84%; (b) N₂H₄.H₂O, EtOH/reflux,10 hours, 92%; (c), (d) 3,5-di-Br-2,4-di-OH-benzaldehyde (1 equivalent),EtOH/reflux, 3 hours, 58%; (e) 2,4-di-SO₃Na-benzaldehyde, DMF/reflux, 4hours, 58%; and (f) 4-sodiumsulfophenyl-isothiocyante, DMF/reflux, 4hours, 47%.

FIG. 10 depicts the reaction scheme for the synthesis of the PEG-ylatedCFTR inhibitor MalH-(PEG)_(n) (Panel A) and MalH-(PEG)_(n)B (Panel B).

FIG. 11 depicts the reaction scheme for the synthesis of the PEG-ylatedCFTR inhibitor GlyH-(PEG)_(n). Reagents and conditions: (i)Br-buterolactone, NaOAc, 90° C., 8 hours, 89%; (j) N₂H₄.H₂O,EtOH/reflux, 10 hours, 89%; (k) (BOC)₂O, THF, rt, 86%; (l) TsCl,pyridine, −15° C., 8 hours, 73%; (m) NH₂-PEG, DMF, 80° C., 24 hours,38%; (n) TFA, CH₂Cl₂, rt 30 min, 73%; and (o)3,5-di-Br-2,4-di-OH-benzaldehyde, EtOH/reflux, 3 hours, 58%.

FIG. 12 is a series of graphs showing inhibition of apical membranechloride current in FRT epithelial cells expressing human wildtype CFTR.Chloride current was measured by short-circuit current analysis in cellssubjected to a chloride ion gradient and after permeabilization of thebasolateral membrane. CFTR was stimulated by 100 μM CPT-cAMP. Increasingconcentrations of MalH compounds were added as indicated.

FIG. 13 is a series of graphs showing intestinal absorption andantidiarrheal efficacy of CFTR inhibitors. Panel A is a graph showingabsorption over 2 hours of indicated MalH compounds in closed jejunalloops in living mice (SD, n=4-6 mice). For comparison absorption ofCFTR_(inh)-172 show as measured by same method. Panel B is a graphshowing inhibition of cholera toxin-induced fluid secretion in closedjejunal loops. Loops were injected with saline (PBS) or salinecontaining 1 μg cholera toxin (CT) with indicated amounts of MalHcompounds. Loops weight-to-length ratio measured at 6 hours (SD, n=3-5mice).

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It should be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aninhibitor” includes a plurality of such inhibitors and reference to “thecell” includes reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of hydrazide-containingcompounds that are high-affinity CFTR inhibitors. The structure of thesecompounds having CFTR inhibitory activity disclosed herein, andderivatives thereof, as well as pharmaceutical formulations and methodsof use are described in more detail below.

DEFINITIONS

A “cystic fibrosis transmembrane conductance regulator protein-mediatedcondition or symptom” or “CFTR-mediated condition or symptom” means anycondition, disorder or disease, or symptom of such condition, disorder,or disease, that results from activity of cystic fibrosis transmembraneconductance regulator protein (CFTR), e.g., activity of CFTR in iontransport. Such conditions, disorders, diseases, or symptoms thereof aretreatable by inhibition of CFTR activity, e.g., inhibition of CFTR iontransport. CFTR activity has been implicated in, for example, intestinalsecretion in response to various agonists, including cholera toxin (see,e.g., Snyder et al. 1982 Bull. World Health Organ. 60:605-613; Chao etal. 1994 EMBO J. 13:1065-1072; Kimberg et al. 1971 J. Clin. Invest.50:1218-1230).

A “CFTR inhibitor” as used herein is a compound that reduces theefficiency of ion transport by CFTR, particularly with respect totransport of chloride ions by CFTR. Preferably CFTR inhibitors of theinvention are specific CFTR inhibitors, i.e., compounds that inhibitCFTR activity without significantly or adversely affecting activity ofother ion transporters, e.g., other chloride transporters, potassiumtransporters, and the like. Preferably the CFTR inhibitors arehigh-affinity CFTR inhibitors, e.g., have an affinity for CFTR of atleast about one micromolar, usually about one to five micromolar.

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature. Preferably, the compound is at leastabout 80%, more preferably at least 90% pure, even more preferably atleast 98% pure, most preferably at least about 99% pure, by weight. Thepresent invention is meant to comprehend diastereomers as well as theirracemic and resolved, enantiomerically pure forms and pharmaceuticallyacceptable salts thereof.

“Treating” or “treatment” as used herein covers the treatment of adisease, condition, disorder or symptom in a subject, wherein thedisease, condition, disorder or symptom is mediated by the activity ofCFTR, and includes: (1) preventing the disease, condition, or disorder,i.e. causing the clinical symptoms of the disease not to develop in asubject that may be exposed to or predisposed to the disease, condition,or disorder, but does not yet experience or display symptoms thereof,(2) inhibiting the disease, condition or disorder, i.e., arresting orreducing the development of the disease, condition or disorder, or itsclinical symptoms, or (3) relieving the disease, condition or disorder,i.e., causing regression of the disease, condition or disorder, or itsclinical symptoms.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound of the invention that, when administered to amammal or other subject in need thereof, is sufficient to effecttreatment, as defined above, for diseases, conditions, disorders orsymptoms mediated by the activity of CFTR. The amount of a compound ofthe invention that constitutes a “therapeutically effective amount” willvary depending on the compound, the disease and its severity and theage, weight, etc., of the subject to be treated, but can be determinedroutinely by one of ordinary skill in the art having regard to his ownknowledge and to this disclosure.

The terms “subject” and “patient” mean a member or members of anymammalian or non-mammalian species that may have a need for thepharmaceutical methods, compositions and treatments described herein.Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), avian, and other subjects. Humans and non-humananimals having commercial importance (e.g., livestock and domesticatedanimals) are of particular interest.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g., primate, murine, lagomorpha, etc.may be used for experimental investigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

A “pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. “A pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and preferably free of contaminants that are capable ofeliciting an undesirable response within the subject. Pharmaceuticalcompositions can be designed for administration to subjects or patientsin need thereof via a number of different routes of administrationincluding oral, buccal, rectal, parenteral, intraperitoneal,intradermal, intracheal and the like. In some embodiments thecomposition is suitable for administration by a transdermal route, usinga penetration enhancer other than DMSO. In other embodiments, thepharmaceutical compositions are suitable for administration by a routeother than transdermal administration.

As used herein, “pharmaceutically acceptable derivatives” of a compoundof the invention include salts, esters, enol ethers, enol esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs thereof. Such derivatives may be readilyprepared by those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

A “pharmaceutically acceptable salt” of a compound of the inventionmeans a salt that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. Such saltsinclude: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

A “pharmaceutically acceptable ester” of a compound of the inventionmeans an ester that is pharmaceutically acceptable and that possessesthe desired pharmacological activity of the parent compound, andincludes, but is not limited to, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl estersof acidic groups, including, but not limited to, carboxylic acids,phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids andboronic acids.

A “pharmaceutically acceptable enol ether” of a compound of theinvention means an enol ether that is pharmaceutically acceptable andthat possesses the desired pharmacological activity of the parentcompound, and includes, but is not limited to, derivatives of formulaC═C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.

A “pharmaceutically acceptable enol ester” of a compound of theinvention means an enol ester that is pharmaceutically acceptable andthat possesses the desired pharmacological activity of the parentcompound, and includes, but is not limited to, derivatives of formulaC═C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.

A “pharmaceutically acceptable solvate or hydrate” of a compound of theinvention means a solvate or hydrate complex that is pharmaceuticallyacceptable and that possesses the desired pharmacological activity ofthe parent compound, and includes, but is not limited to, complexes of acompound of the invention with one or more solvent or water molecules,or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solventor water molecules.

A “pro-drug” means any compound that releases an active parent compoundof formula (I) in vivo when the prodrug is administered to a mammaliansubject. Prodrugs of the compounds of formula (I) contain functionalgroups that, under standard physiological conditions, are hydrolyzedinto the corresponding carboxy, hydroxy, or amino group. Examples ofsuch functional groups include, but are not limited to, esters (e.g.,acetate, formate and benzoate derivatives) and carbamates (e.g.,N,N-dimethylaminocarbonyl) of hydroxy groups in compounds of formula(I), and the like. Additional examples include dipeptide or tripeptideesters of hydroxy or carboxy groups in compounds of formula (I), and thelike. The preparation of such functional groups is well known in theart. For example, a compound of formula (I) having a hydroxy groupattached thereto may be treated with a carboxylic acid or a dipeptidehaving a free carboxy terminus under esterification conditions wellknown in the art to yield the desired ester functional group. Likewise,a compound of formula (I) having a free carboxy group attached theretomay be treated with an alcohol or a tripeptide containing a hydroxygroup such as a serine residue (e.g., —N(H)—C(H)(CH₂OH)—C(O)—) underesterification conditions well known in the art to produce the desiredester functional group. In addition, compounds of formula (I) having acarboxylic ester group attached thereto may be treated with a differentcarboxylic ester under standard transesterification conditions toproduce compounds of formula (I) with the desired functional ester groupattached thereto. All such functional groups are considered to be withinthe scope of this invention.

The term “organic group” and “organic radical” as used herein means anycarbon-containing group, including hydrocarbon groups that areclassified as an aliphatic group, cyclic group, aromatic group,functionalized derivatives thereof and/or various combination thereof.The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, andthe like. Suitable substituents include carboxy, protected carboxy,amino, protected amino, halo, hydroxy, protected hydroxy, mercapto,lower alkylthio, nitro, cyano, monosubstituted amino, protectedmonosubstituted amino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇acyl, C₁ to C₇ acyloxy, and the like. The term “substituted alkyl” meansthe above defined alkyl group substituted from one to three times by ahydroxy, protected hydroxy, amino, protected amino, cyano, halo,trifloromethyl, mono-substituted amino, di-substituted amino, loweralkoxy, mercapto, lower alkylthio, carboxy, protected carboxy, or acarboxy, amino, and/or hydroxy salt. As used in conjunction with thesubstituents for the heteroaryl rings, the terms “substituted(cycloalkyl)alkyl” and “substituted cycloalkyl” are as defined belowsubstituted with the same groups as listed for a “substituted alkyl”group. The term “alkenyl group” means an unsaturated, linear or branchedhydrocarbon group with one or more carbon-carbon double bonds, such as avinyl group. The term “alkynyl group” means an unsaturated, linear orbranched hydrocarbon group with one or more carbon-carbon triple bonds.The term “cyclic group” means a closed ring hydrocarbon group that isclassified as an alicyclic group, aromatic group, or heterocyclic group.The term “alicyclic group” means a cyclic hydrocarbon group havingproperties resembling those of aliphatic groups. The term “aromaticgroup” or “aryl group” means a mono- or polycyclic aromatic hydrocarbongroup, and may include one or more heteroatoms, and which are furtherdefined below. The term “heterocyclic group” means a closed ringhydrocarbon in which one or more of the atoms in the ring are an elementother than carbon (e.g., nitrogen, oxygen, sulfur, etc.), and arefurther defined below.

“Organic groups” may be functionalized or otherwise comprise additionalfunctionalities associated with the organic group, such as carboxyl,amino, hydroxyl, and the like, which may be protected or unprotected.For example, the phrase “alkyl group” is intended to include not onlypure open chain saturated hydrocarbon alkyl substituents, such asmethyl, ethyl, propyl, t-butyl, and the like, but also alkylsubstituents bearing further substituents known in the art, such ashydroxy, alkoxy, mercapto, alkylthio, alkylsulfonyl, halo, cyano, nitro,amino, carboxyl, etc. Thus, “alkyl group” includes ethers, esters,haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms “halo group” or “halogen” are used interchangeably herein andrefer to the fluoro, chloro, bromo or iodo groups.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans-decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, mercapto, alkylthio, trifluoromethyl, C₁ to C₇alkyl, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy,oxycarboxy, protected carboxy, carboxymethyl, protected carboxymethyl,hydroxymethyl, protected hydroxymethyl, amino, protected amino,(monosubstituted)amino, protected (monosubstituted)amino,(disubstituted)amino, carboxamide, protected carboxamide, N—(C₁ to C₆alkyl)carboxamide, protected N—(C₁ to C₆ alkyl)carboxamide, N,N-di(C₁ toC₆ alkyl)carboxamide, trifluoromethyl, N—((C₁ to C₆alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl, substituted orunsubstituted, such that, for example, a biphenyl or naphthyl groupresults.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2-, 3- or 4-chlorophenyl,2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2-, 3- or4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-, 3- or4-fluorophenyl and the like; a mono or di(hydroxy)phenyl group such as2, 3, or 4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxyderivatives thereof and the like; a nitrophenyl group such as 2-, 3- or4-nitrophenyl; a cyanophenyl group, for example, 2-, 3- or4-cyanophenyl; a mono- or di(alkyl)phenyl group such as 2-, 3- or4-methylphenyl, 2,4-dimethylphenyl, 2-, 3- or 4-(iso-propyl)phenyl, 2-,3- or 4-ethylphenyl, 2-, 3- or 4-(n-propyl)phenyl and the like; a monoor di(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2-, 3- or4-(isopropoxy)phenyl, 2-, 3- or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; a mono- or di(halo)-, mono-, di-or tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and3-bromo-4-hydroxyphenyl and the like; a mono- or di(halo)-mono- ordi-(hydroxyl)-mono- or di-(alkoxy)phenyl such as3,5-dibromo-2-hydroxyl-4-methoxyphenyl and the like; 2-, 3- or4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protectedcarboxy)phenyl group such as 2-, 3- or 4-carboxyphenyl or2,4-di(protected carboxy)phenyl; a mono- or di(hydroxymethyl)phenyl or(protected hydroxymethyl)phenyl such as 2-, 3- or 4-(protectedhydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- ordi(aminomethyl)phenyl or (protected aminomethyl)phenyl such as 2-, 3- or4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono-or di(N-(methylsulfonylamino))phenyl such as 2-, 3- or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)-n-hexyl,2-(5′-cyano-3′-methoxyphenyl)-n-pentyl, 3-(2′,6′-dimethylphenyl)propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxyhexyl,5-(4′-aminomethylphenyl)-3-(aminomethyl)pentyl, 5-phenyl-3-oxopent-1-yl,(4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to five and sixmembered carbocyclic rings. Also as noted above, the term “heteroaryl”denotes optionally substituted five-membered or six-membered rings thathave 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms,in particular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms. These five-membered or six-membered rings may befully unsaturated.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, and preferably to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, mercapto, lower alkylthio, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,(cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substitutedphenyl, phenylalkyl, and (substituted phenyl)alkyl. Substituents for theheteroaryl group are as heretofore defined, or in the case oftrihalomethyl, can be trifluoromethyl, trichloromethyl, tribromomethyl,or triiodomethyl. As used in conjunction with the above substituents forheteroaryl rings, “lower alkoxy” means a C₁ to C₄ alkoxy group,similarly, “lower alkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ toC₇ substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or disubstituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

The term “electron-withdrawing group” refers to the ability of afunctional group on a molecule to draw electrons to it self more than ahydrogen atom would if the hydrogen atom occupied the same position inthe molecule. Examples of electron-withdrawing groups include, but arenot limited to, halogen groups, —C(O)R groups (where R is alkyl);carboxylic acid and ester groups; —NR3+groups (where R is alkyl orhydrogen); azo; nitro; —OR and —SR groups (where R is hydrogen oralkyl); and organic groups (as defined herein) containing suchelectron-withdrawing groups, such as haloalkyl groups (includingperhaloalkyl groups), and the like.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either an individual enantiomer or as a mixture ofthereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

Overview

The invention provides hydrazide-containing compounds, derivativecompositions and methods of their use in high affinity inhibition ofcystic fibrosis transmembrane conductance regulator protein (CFTR) andfor the study and treatment of CFTR-mediated diseases and conditions.The discovery of the subject hydrazide-containing compounds andderivatives was based on screening of numerous potential candidatecompounds using an assay designed to identify CFTR inhibitors thatinteract directly with CFTR. Without being held to any particular theoryor mode of operation, since multiple CFTR activators that work ondifferent activating pathways were included in the studies leading toidentification of the subject compounds, the inhibitory compounds of theinvention likely effect inhibition by acting at or near the CFTR Cl⁻transporting pathway. A screening of 100,000 diverse compoundsidentified several compounds and derivatives as effective CFTRinhibitors (FIG. 1B). These compounds and derivatives are unrelatedchemically and structurally to previously known CFTR activators or tothe previously known CFTR inhibitors DPC, NPPB glibenclamide, orthiazolidinone. The most potent CFTR inhibitor identified from screeninghad a K_(I) of ˜2 μM for inhibition of Cl⁻ current in human airwaycells. Inhibition was rapid, reversible and CFTR-specific.

The compositions and methods of the invention will now be described inmore detail.

Hydrazide-Containing Compounds

The hydrazide-containing compounds described herein comprise anaromatic- or heteroaromatic-substituted nitrogen, a hydrazide (which canbe a glycine or oxamic hydrazide), and a substituted or substituted arylgroup. In specific embodiments, the subject compounds are generallydescribed by Formula (I) as follows:

wherein X is independently chosen from an alkyl group, or a carbonylgroup; Y is independently chosen from an alky group; an alkyl grouphaving polar substitutions, such as a sulfo group, or a carboxyl group,or a linker, such as an amide bond or an ether linker to provide forattachment of one or more larger polar molecules, such as a polyoxyalkylpolyether (such as a polyethylene glycol (PEG), polypropylene glycol,polyhydroxyethyl glycerol), disaccharides, a substituted orunsubstituted phenyl group, polyalkylimines, a dendrimer from 0-10generation and the like, where Y can further include such an attachedpolar molecule(s); R₁ is independently chosen from a substituted orunsubstituted phenyl group, a substituted or unsubstitutedheteroaromatic group such as a substituted or unsubstituted quinolinylgroup, an substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenand an alkyl group; or a pharmaceutically acceptable derivative thereof,as an individual stereoisomer or a mixture thereof. In one embodiment,R₁ is chosen from a substituted phenyl group, an unsubstitutedquinolinyl group, an unsubstituted anthracenyl group, and anunsubstituted naphthalenyl group; R₂ is a substituted phenyl group; andR₃ is independently chosen from hydrogen and an alkyl group. Exemplarysubstituents for R₁, R₂, and R₃, are described in more detail below.

In certain embodiments, the hydrazide-containing compounds are generallydescribed by Formula (I), wherein X is an alkyl group. Such compoundsare generally described by Formula (Ia) as follows:

wherein Y is a hydrogen or an alkyl group such as a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, e.g., methyl, ethyl, isopropyl, tert-butyl,heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl; X₁ isindependently chosen from a hydrogen or an alkyl group such as asubstituted or unsubstituted, saturated linear or branched hydrocarbongroup or chain (e.g., C₁ to C₈) including, e.g., methyl, ethyl,isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, or an alkyl group comprising a polar molecule chosen froma sulfo group, a carboxy group, a carboxamide group, a polyoxyalkylpolyether, a disaccharide, a substitute or unsubstituted phenyl group,or a polyethylene imine (PEI), or a dendrimer from 0-10 generation; R₁is independently chosen from a substituted or unsubstituted phenylgroup, a substituted or unsubstituted heteroaromatic group such as aquinolinyl group, a substituted or unsubstituted anthracenyl group, anda substituted or unsubstituted naphthalenyl group; R₂ is a substitutedor unsubstituted phenyl group; and R₃ is independently chosen fromhydrogen and an alkyl group. In some embodiments, when X₁ is hydrogen,R₁ is a substituted or unsubstituted anthracenyl group, or aheteroaromatic group. In still other embodiments, when X₁ is hydrogen, Yis not hydrogen.

In specific embodiments, R₁ is independently chosen from amono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; amono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a naphthalenylgroup such as 1- or 2-naphthalenyl; a mono- or di(halo)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-chloronaphthalenyl, 3,4- or 5,6- or5,7- or 5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,dihydroxynaphthalenyl; a mono- or di or tri(alkoxy)naphthalenyl, such as1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl, 5,8-dimethoxynaphthalenyl,1,4,8-trimethoxynaphthalenyl; a mono- or di(alkyl)naphthalenyl, such as1-, 3-, 4-, 5-, or 6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl;a mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as1methyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as6-quinolynyl; R₂ is independently chosen from the group consisting ofsubstituted phenyl groups such as: a mono-(halo)phenyl group such as 2-,3-, or 4-bromophenyl; a mono or di(hydroxyl)phenyl group such as2,3,4-hydroxyphenyl and 2,4-dihydroxyphenyl; a mono- or di(halo)-mono-,di-, or tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- ordi-(hydroxyl)-mono- or di-(alkoxy)phenyl such as3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R₃ is independently chosenfrom hydrogen or an alkyl group.

In further embodiments, the hydrazide-containing compounds andderivatives of Formula (Ia) may comprise of compounds, wherein Y is ahydrogen; X is a hydrogen, methyl or ethyl group; R₁ is independentlychosen from a mono-(halo)phenyl group, such as a 2-, 3-, or4-chlorophenyl group, a naphthalenyl group, such as a 2-naphthalenyl ora 1-naphthalenyl; R₂ is independently chosen from a di-(halo)-mono- ordi(hydroxyl)phenyl group such as a 3,5-di-bromo-2,4-di-hydroxyphenylgroup, 3,5-di-bromo-4-hydroxyphenyl group; and R₃ is a hydrogen or amethyl group.

In other embodiments, the hydrazide-containing compounds are generallydescribed by Formula (I) wherein X is CH₂. Such compounds are generallydescribed by Formula (Ib) as follows:

wherein Y is a hydrogen or an alkyl group such as a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, e.g., methyl, ethyl, isopropyl, tert-butyl,heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl; R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenand an alkyl group.

In some embodiments, Y is an alkyl group such as a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, e.g., methyl, ethyl, isopropyl, tert-butyl,heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl; and R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenand an alkyl group.

In specific embodiments, R₁ is independently chosen from amono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; amono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a naphthalenylgroup such as 1- or 2-naphthalenyl; a mono- or di(halo)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-chloronaphthalenyl, 3,4- or 5,6- or5,7- or 5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,dihydroxynaphthalenyl; a mono- or di or tri(alkoxy)naphthalenyl, such as1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl, 5,8-dimethoxynaphthalenyl,1,4,8-trimethoxynaphthalenyl; a mono- or di(alkyl)naphthalenyl, such as1-, 3-, 4-, 5-, or 6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl;a mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as1methyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as6-quinolynyl; R₂ is independently chosen from the group consisting ofsubstituted phenyl groups such as: a mono-(halo)phenyl group such as 2-,3-, or 4-bromophenyl; a mono or di(hydroxyl)phenyl group such as2,3,4-hydroxyphenyl and 2,4-dihydroxyphenyl; a mono- or di(halo)-mono-,di-, or tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- ordi-(hydroxyl)-mono- or di-(alkoxy)phenyl such as3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R₃ is independently chosenfrom hydrogen or an alkyl group. Compounds described by Formula (Ib) aregenerally described as glycine hydrazides.

In further embodiments, the hydrazide-containing compounds andderivatives of Formula (Ib) may comprise of compounds, wherein Y is ahydrogen; R₁ is independently chosen from a mono-(halo)phenyl group,such as a 2-, 3-, or 4-chlorophenyl group, a naphthalenyl group, such asa 2-naphthalenyl or a 1-naphthalenyl; R₂ is independently chosen from adi-(halo)-mono- or di(hydroxyl)phenyl group such as a3,5-di-bromo-2,4-di-hydroxyphenyl group, 3,5-di-bromo-4-hydroxyphenylgroup; and R₃ is a hydrogen or a methyl group.

In yet other embodiments, the hydrazide-containing compounds aregenerally described by Formula (I) wherein X is a carbonyl. Suchcompounds are generally described by Formula (Ic) as follows:

wherein Y is a hydrogen or an alkyl group such as a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, e.g., methyl, ethyl, isopropyl, tert-butyl,heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl; R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenand an alkyl group.

In some embodiments, Y is an alkyl group such as a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, e.g., methyl, ethyl, isopropyl, tert-butyl,heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl; R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenand an alkyl group.

In specific embodiments, R₁ is independently chosen from amono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; amono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a naphthalenylgroup such as 1- or 2-naphthalenyl; a mono- or di(halo)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-chloronaphthalenyl, 3,4- or 5,6- or5,7- or 5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,dihydroxynaphthalenyl; a mono- or di or tri(alkoxy)naphthalenyl, such as1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl, 5,8-dimethoxynaphthalenyl,1,4,8-trimethoxynaphthalenyl; a mono- or di(alkyl)naphthalenyl, such as1-, 3-, 4-, 5-, or 6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl;a mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as1methyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as6-quinolynyl; R₂ is independently chosen from the group consisting ofsubstituted phenyl groups such as: a mono-(halo)phenyl group such as 2-,3-, or 4-bromophenyl; a mono or di(hydroxyl)phenyl group such as2,3,4-hydroxyphenyl and 2,4-dihydroxyphenyl; a mono- or di(halo)-mono-,di-, or tri-(hydroxyl)phenyl such as 3,5-dibromo-2,4,6-trihydroxyphenyl,3,5-dibromo-2,4-dihydroxyphenyl, 3,5-dibromo-4-hydroxyphenyl, and3-bromo-4-hydroxyphenyl; a mono- or di(halo)-mono- ordi-(hydroxyl)-mono- or di-(alkoxy)phenyl such as3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R₃ is independently chosenfrom hydrogen or an alkyl group. Compounds described by Formula (Ic) aregenerally described as oxamic acid hydrazides.

In further embodiments, the hydrazide-containing compounds andderivatives of Formula (Ic) may comprise of compounds, wherein Y ishydrogen; R₁ is a naphthalenyl group, such as a 2-naphthalenyl or a1-naphthalenyl; R₂ is a di-(halo)-mono- or di(hydroxyl)phenyl group suchas a 3,5-di-bromo-2,4-di-hydroxyphenyl group,3,5-di-bromo-4-hydroxyphenyl group; and R₃ is a hydrogen or a methylgroup.

In some embodiments of the invention, the hydrazide-containing compoundmay comprise a formula of the following:

The hydrazide-containing compounds described herein may be modified, forexample, to provide for a desired characteristic. Preferably,modification of the compounds does not significantly or undesirablyadversely affect the desirable characteristics of thehydrazide-containing compounds, e.g., ability to inhibit CFTR functionand water solubility of the compound. For example, the compoundsdescribed herein can be modified so as decrease the ability of thecompound to cross a cell membrane, e.g., a cell membrane of a celllining a mucosal surface, e.g., a gastrointestinal cell. Membraneimpermeance of the compounds disclosed herein can be increased by, forexample, increasing the size or other physical characteristics of thecompound.

In such embodiments, the membrane permeability of the compoundsgenerally described by Formula I, are decreased by the addition of polargroups, such as sulfo and alkyl-carboxyl groups. Such compounds aregenerally described by Formula (I) as follows:

wherein Y is independently chosen from an alky group; an alkyl grouphaving polar substitutions, such as a sulfo group, or a carboxyl group;or a linker, such as an amide bond or an ether linker to provide forattachment of one or more larger polar molecules, such as a polyoxyalkylpolyether (such as a polyethylene glycol (PEG), polypropylene glycol,polyhydroxyethyl glycerol), disaccharides, polyalkylimines, and thelike, where Y can further include such an attached polar molecule(s); Xis independently chosen from an alkyl group, or a carbonyl group; R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenand an alkyl group.

In specific embodiments Y is independently chosen from a substituted orunsubstituted alkyl group, such as a substituted or unsubstituted,saturated linear or branched hydrocarbon group or chain (e.g., C₁ to C₈)including, e.g., methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl,dodecyl, octadecyl, amyl, 2-ethylhexyl; an alkyl group carrying polargroups such as hydroxy, sulfo, carboxylate, or a substituted orunsubstituted carboxamide groups (where exemplary groups include3-sulfopropyl, 4-sulfobutyl, carboxymethyl, 2-carboxypropyl,2-methoxy-2-oxoethyl, 3-methoxy-3-oxopropyl); or a linker such as anamide bond or ether linker to provide for attachment of one or morelarger polar molecules, such as a polyoxyalkyl polyether (such aspolyethylene glycol (PEG), polypropylene glycol, polyhydroxyethylglycerol), polyethyleneimines, disaccharides, trisaccharides,polyalkylimines, small amino dextrans and the like, where Y can furtherinclude such an attached polar molecule(s).

In some embodiments, the nitrogen of the unsaturated amide bond of thecompound may be substituted as exemplified below:

wherein X is independently chosen from an alkyl group, or a carbonylgroup; R₁ is independently chosen from a substituted or unsubstitutedphenyl group, a substituted or unsubstituted heteroaromatic group suchas a quinolinyl group, a substituted or unsubstituted anthracenyl group,and a substituted or unsubstituted naphthalenyl group; R₂ is asubstituted or unsubstituted phenyl group; R₃ is independently chosenfrom hydrogen and an alkyl group; and Y′ is independently chosen from ansubstituted or unsubstituted alkyl group, such as a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, e.g., methyl, ethyl, isopropyl, tert-butyl,heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl; an alkyl groupcarrying polar groups such as hydroxy, sulfo, carboxylate, and asubstituted or unsubstituted carboxamide groups (where exemplary groupsinclude, such as 3-sulfopropyl, 4-sulfobutyl, carboxymethyl,2-carboxypropyl, 2-methoxy-2-oxoethyl, 3-methoxy-3-oxoproplyl); or alinker such as an amide bond or ether linker to provide for attachmentof one or more to larger polar molecules, such as a polyoxyalkylpolyether (such as polyethylene glycol (PEG), polypropylene glycol,polyhydroxyethyl glycerol), polyethyleneimines, disaccharides,trisaccharides, polyalkylimines, small amino dextrans and the like,where Y′ can further include such an attached polar molecule(s).

In some embodiments, X is independently chosen from a carbonyl group; analkyl group, such as a substituted or unsubstituted, saturated linear orbranched hydrocarbon group or chain (e.g., C₁ to C₈) including,methylene, substituted alkyl groups, such as propene; substituted orunsubstituted phenyl groups, such as a phenyl group carrying polargroups; or a linker to carry polar groups; R₁ is independently chosenfrom a mono-(halo)phenyl group such as 2-, 3-, or 4-chlorophenyl; amono-(alkyl)phenyl such as a 2-, 3-, or 4-methylphenyl; a naphthalenylgroup such as 1- or 2-naphthalenyl; a mono- or di(halo)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-chloronaphthalenyl, 3,4- or 5,6- or5,7- or 5,8-dichloronaphthalenyl; a mono- or di(hydroxy)naphthalenyl,such as 1-, 3-, 4-, 5-, 6-, 7-, or 8-hydroxynaphthalenyl, 1,8-, 3,4-,dihydroxynaphthalenyl; a mono- or di or tri(alkoxy)naphthalenyl, such as1-, 3-, 5-, 6-, 7-, or 8-methoxynaphthalenyl, 5,8-dimethoxynaphthalenyl,1,4,8-trimethoxynaphthalenyl; a mono- or di(alkyl)naphthalenyl, such as1-, 3-, 4-, 5-, or 6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl;a mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as1methyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as6-quinolynyl; R₂ is independently chosen from the group consisting ofsubstituted phenyl groups such as a mono-(halo)phenyl group such as 2-,3-, or 4-bromophenyl; a mono or di(hydroxyl)phenyl group such as2,3,4-hydroxyphenyl and 2,4-dihydroxyphenyl; a mono- or di(halo)-mono-or di- or tri-(hydroxyl)phenyl such as3,5-dibromo-2,4,6-trihydroxyphenyl, 3,5-dibromo-2,4-dihydroxyphenyl,3,5-dibromo-4-hydroxyphenyl, and 3-bromo-4-hydroxyphenyl; a mono- ordi(halo)-mono- or di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as3,5-dibromo-2-hydroxy-4-methoxyphenyl; and R₃ is independently chosenfrom hydrogen or an alkyl group.

In some embodiments, X of the compound may be substituted as exemplifiedbelow:

wherein X is an alkyl group; R₁ is independently chosen from asubstituted or unsubstituted phenyl group, a substituted orunsubstituted heteroaromatic group such as a quinolinyl group, asubstituted or unsubstituted anthracenyl group, and a substituted orunsubstituted naphthalenyl group; R₂ is a substituted or unsubstitutedphenyl group; R₃ is independently chosen from hydrogen and an alkylgroup; and Y″ is independently chosen from an substituted orunsubstituted alkyl group, such as a substituted or unsubstituted,saturated linear or branched hydrocarbon group or chain (e.g., C₁ to C₈)including, e.g., methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl,dodecyl, octadecyl, amyl, 2-ethylhexyl; an alkyl group carrying polargroups such as hydroxy, sulfo, carboxylate, and a substituted orunsubstituted carboxamide groups (where exemplary groups include, suchas 3-sulfopropyl, 4-sulfobutyl, carboxymethyl, 2-carboxypropyl,2-methoxy-2-oxoethyl, 3-methoxy-3-oxoproplyl); or a linker such as anamide bond or ether linker to provide for attachment of one or more tolarger polar molecules, such as substituted or unsubstituted phenylgroup, a polyoxyalkyl polyether (such as polyethylene glycol (PEG),polypropylene glycol, polyhydroxyethyl glycerol), polyethyleneimines,disaccharides, trisaccharides, polyalkylimines, small amino dextrans, adendrimer from 0-10 generation, and the like, where Y″ can furtherinclude such an attached polar molecule(s).

In some embodiments, X is a substituted alkyl group, such as a methylgroup carrying polar groups or a linker to carry polar groups; R₁ isindependently chosen from a mono-(halo)phenyl group such as 2-, 3-, or4-chlorophenyl; a mono-(alkyl)phenyl such as a 2-, 3-, or4-methylphenyl; a naphthalenyl group such as 1- or 2-naphthalenyl; amono- or di(halo)naphthalenyl, such as 1-, 3-, 4-, 5-, 6-, 7-, or8-chloronaphthalenyl, 3,4- or 5,6- or 5,7- or 5,8-dichloronaphthalenyl;a mono- or di(hydroxy)naphthalenyl, such as 1-, 3-, 4-, 5-, 6-, 7-, or8-hydroxynaphthalenyl, 1,8-, 3,4-, dihydroxynaphthalenyl; a mono- or dior tri(alkoxy)naphthalenyl, such as 1-, 3-, 5-, 6-, 7-, or8-methoxynaphthalenyl, 5,8-dimethoxynaphthalenyl,1,4,8-trimethoxynaphthalenyl; a mono- or di(alkyl)naphthalenyl, such as1-, 3-, 4-, 5-, or 6-methylnaphthalenyl, 4,5-, 4,6-dimethynaphthalenyl;a mono-(hydroxy)-mono or di(sulfo)naphthalenyl such as4-hydroxy-2-sulfo-naphthalenyl, 8-hydroxy-3,6-disulfo-naphthalenyl;mono(alkyl)-mono- or di(alkoxy)naphthalenyl, such as1methyl-5,6-dimethoxynaphthalenyl; or a quinolinyl group such as6-quinolynyl; R₂ is independently chosen from the group consisting ofsubstituted phenyl groups such as a mono-(halo)phenyl group such as 2-,3-, or 4-bromophenyl; a mono or di(hydroxyl)phenyl group such as2,3,4-hydroxyphenyl and 2,4-dihydroxyphenyl; a mono- or di(halo)-mono-or di- or tri-(hydroxyl)phenyl such as3,5-dibromo-2,4,6-trihydroxyphenyl, 3,5-dibromo-2,4-dihydroxyphenyl,3,5-dibromo-4-hydroxyphenyl, and 3-bromo-4-hydroxyphenyl; a mono- ordi(halo)-mono- or di-(hydroxyl)-mono- or di-(alkoxy)phenyl such as3,5-dibromo-2-hydroxy-4-methoxyphenyl; R₃ is independently chosen fromhydrogen or an alkyl group; and Y″ is independently chosen from an alkygroup; an alkyl group having polar substitutions, such as a sulfo group,or a carboxyl group; or a linker, such as an amide bond or an etherlinker, to provide for attachment of one or more larger polar molecules,a polyoxyalkyl polyether (such as polyethylene glycol (PEG),polypropylene glycol, polyhydroxyethyl glycerol), disaccharides,polyalkylimines, and a substituted or unsubstituted phenyl group, suchas a 2,4-dihydroxy-3,5-di-bromophenyl group, a2,4-disodium-disulfophenyl group, and a 3-monosodium-monosulfophenylgroup, where Y can further include such an attached polar molecule(s).

In some embodiments of the invention, the hydrazide-containing compoundmay comprise a formula of the following:

In further embodiments, the hydrazide containing compounds are dimerizedby using a bifunctional linker with varied chain lengths. Such compoundsare cell impermeant due to their large, bulky nature and sterichindrance. In specific embodiments, the subject compounds are generallydescribed by Formula (Id) as follows:

wherein Z is a monomeric or polymeric unit, such as a polyoxyalkylpolyether (such as a polyethylene glycol, polypropylene glycol,polyhydroxyethyl glycerol), a linear polyamine, or a bifunctionalpolysaccharide; and n is in the range of 0 to 500, 1 to 450, 2 to 400, 5to 300, 10 to 250, 20 to 200, 30 to 150, 40 to 100, 50 to 90, and thelike. In certain embodiments N has a range of 0 to 100, 1 to 95, 10 to90, 20 to 80, 30 to 70, 40 to 60, and the like. In specific embodiments,X is independently chosen from an alkyl group, or a carbonyl group; Y isindependently chosen from an alky group; an alkyl group having polarsubstitutions, such as a sulfo group, or a carboxyl group; or a linker,such as an amide bond or an ether linker, to provide for attachment ofone or more larger polar molecules, a polyoxyalkyl polyether (such aspolyethylene glycol (PEG), polypropylene glycol, polyhydroxyethylglycerol), disaccharides, polyalkylimines, and the like, where Y canfurther include such an attached polar molecule(s); R₁, is independentlychosen from a substituted phenyl group, a quinolinyl group, ananthracenyl group, and a naphthalenyl group; R₂ is a substituted phenylgroup; and R₃ is independently chosen from hydrogen and an alkyl group;or a pharmaceutically acceptable derivative thereof, as an individualstereoisomer or a mixture thereof.

In some embodiments of the invention, the hydrazide-containing compoundmay comprise a formula of the following:

Pharmaceutical Preparations

Also provided by the invention are pharmaceutical preparations of thesubject hydrazide-containing compounds described above. The subjectcompounds can be incorporated into a variety of formulations fortherapeutic administration by a variety of routes. More particularly,the compounds of the present invention can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers, diluents, excipients and/oradjuvants, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.Preferably, the formulations are free of detectable DMSO (dimethylsulfoxide), or are formulated with a penetration enhancer other thanDMSO. The formulations may be designed for administration to subjects orpatients in need thereof via a number of different routes, which may beparenteral or enteral. Exemplary routes of administration include oral,buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal,intracheal, etc., administration.

In pharmaceutical dosage forms, the subject compounds of the inventionmay be administered in the form of their pharmaceutically acceptablederivative, such as a salt, or they may also be used alone or inappropriate association, as well as in combination, with otherpharmaceutically active compounds. The following methods and excipientsare merely exemplary and are in no way limiting.

In one embodiment of particular interest, the compounds of the inventionare administered to the gastrointestinal tract of the subject, so as toprovide for decreased fluid secretion. Suitable formulations for thisembodiment of the invention include any formulation that provides fordelivery of the compound to the gastrointestinal surface, particularlyan intestinal tract surface.

For oral formulations, the subject compounds can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such asstarch, gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth, orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes,crystalline cellulose, cellulose derivatives, and acacia; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose, methyl cellulose, agar, bentonite, or xanthangum; with lubricants, such as talc, sodium oleate, magnesium stearatesodium stearate, sodium benzoate, sodium acetate, or sodium chloride;and if desired, with diluents, buffering agents, moistening agents,preservatives, coloring agents, and flavoring agents. Of particularinterest is formulation of the subject hydrazide-containing compoundswith a buffering agent, to provide for protection of the compound fromlow pH of the gastric environment. It may also be preferable to providean enteric coating. In one embodiment, the compounds are formulated fororal delivery with a flavoring agent, e.g., in a liquid, solid orsemi-solid formulation.

Oral formulations can be provided as gelatin capsules, which may containthe active substance and powdered carriers, such as lactose, starch,cellulose derivatives, magnesium stearate, stearic acid, and the like.Similar carriers and diluents may be used to make compressed tablets.Tablets and capsules can be manufactured as sustained release productsto provide for continuous release of active ingredients over a period oftime. Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, or entericcoated for selective disintegration in the gastrointestinal tract.Liquid dosage forms for oral administration may contain coloring and/orflavoring agents to increase patient acceptance.

Other suitable oral formulations include those that provide forsustained release, which may be controlled release, of the compound.Such formulations include hydrogels, microparticles, and other dosageforms and formulations known in the art.

Water, a suitable oil, saline, aqueous dextrose, and related sugarsolutions and glycols such as propylene glycol or polyethylene glycols,may be used as carriers for parenteral solutions. Such solutions canalso contain a water soluble salt of the active ingredient, suitablestabilizing agents, and if necessary, buffer substances. Suitablestabilizing agents include antioxidizing agents such as sodiumbisulfate, sodium sulfite, or ascorbic acid, either alone or combined,citric acid and its salts and sodium EDTA. Parenteral solutions may alsocontain preservatives, such as benzalkonium chloride, methyl- orpropyl-paraben, and chlorobutanol.

The subject compounds of the invention can be formulated intopreparations for injection by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives. Where desired, solubilizers foruse can include vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000succinate), cyclodextrins, and the like.

Furthermore, the subject compounds can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. The compounds of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The compounds of the invention can be utilized in aerosol formulation tobe administered via inhalation. The compounds of the present inventioncan be formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

In one embodiment, topical administration (e.g., by transdermaladministration) is of interest. Topical formulations can be in the formof a transdermal patch, ointment, paste, lotion, cream, gel, and thelike. Topical formulations may include one or more of a penetratingagent, thickener, diluent, emulsifier, dispersing aid, or binder. Wherethe compound is formulated for transdermal delivery, the compound may beformulated with or for use with a penetration enhancer. Penetrationenhancers, which include chemical penetration enhancers and physicalpenetration enhancers, facilitate delivery of the compound through theskin, and may also be referred to as “permeation enhancers”interchangeably. Physical penetration enhancers include, for example,electrophoretic techniques such as iontophoresis, use of ultrasound (or“phonophoresis”), and the like. Chemical penetration enhancers areagents administered either prior to, with, or immediately followingcompound administration, which increase the permeability of the skin,particularly the stratum corneum, to provide for enhanced penetration ofthe drug through the skin.

Compounds that have been used to enhance skin permeability include: thesulfoxides dimethylsulfoxide (DMSO) and decylmethylsulfoxide (C₁₀ MSO);ethers such as diethylene glycol monoethyl ether,dekaoxyethylene-oleylether, and diethylene glycol monomethyl ether;surfactants such as sodium laurate, sodium lauryl sulfate,cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer (231,182, 184), Tween (20, 40, 60, 80) and lecithin; the 1-substitutedazacycloheptan-2-ones, particularly 1-n-dodecylcyclazacycloheptan-2-one;alcohols such as ethanol, propanol, octanol, benzyl alcohol, and thelike; petrolatums, such as petroleum jelly (petrolatum), mineral oil(liquid petrolatum), and the like; fatty acids such as C₈-C₂₂ and otherfatty acids (e.g., isostearic acid, octanoic acid, oleic acid, lauricacid, valeric acid); C₈-C₂₂ fatty alcohols (e.g., oleyl alcohol, laurylalcohol); lower alkyl esters of C₈-C₂₂ fatty acids and other fatty acids(e.g., ethyl oleate, isopropyl myristate, butyl stearate, methyllaurate, isopropyl myristate, isopropyl palmitate, methylpropionate,ethyl oleate); monoglycerides of C₈-C₂₂ fatty acids (e.g., glycerylmonolaurate); tetrahydrofurfuryl alcohol polyethylene glycol ether;2-(2-ethoxyethoxy)ethanol; diethylene glycol monomethyl ether; alkylarylethers of polyethylene oxide; polyethylene oxide monomethyl ethers;polyethylene oxide dimethyl ethers; di-lower alkyl esters of C₆-C₈diacids (e.g., diisopropyl adipate); ethyl acetate; acetoacetic ester;polyols and esters thereof such as propylene glycol, ethylene glycol,glycerol, butanediol, polyethylene glycol, and polyethylene glycolmonolaurate; amides and other nitrogenous compounds such as urea,dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone,N-alkylpyrrolidone, e.g., 1-methyl-2-pyrrolidone; ethanol amine,diethanol amine and triethanolamine; terpenes; alkanones, and organicacids, particularly salicylic acid and salicylates, citric acid andsuccinic acid. Additional chemical and physical penetration enhancersare described in, for example, Transdermal Delivery of Drugs, A. F.Kydonieus (ED) 1987 CRL Press; Percutaneous Penetration Enhancers, eds.Smith et al. (CRC Press, 1995); Lenneruas et al., J Pharm. Pharmacol.2002; 54(4):499-508; Karande et al., Pharm. Res. 2002; 19(5):655-60;Vaddi et al., J. Pharm. Sci. 2002 July; 91(7):1639-51; Ventura et al.,J. Drug Target 2001; 9(5):379-93; Shokri et al., Int. J. Pharm. 2001;228(1-2):99-107; Suzuki et al., Biol. Pharm. Bull. 2001; 24(6):698-700;Alberti et al., J. Control Release 2001; 71(3):319-27; Goldstein et al.,Urology 2001; 57(2):301-5; Kiijavainen et al., Eur. J. Pharm. Sci. 2000;10(2):97-102; and Tenjarla et al., Int. J. Pharm. 1999; 192(2):147-58.

Where the compound is formulated with a chemical penetration enhancer,the penetration enhancer is selected for compatibility with thecompound, and is present in an amount sufficient to facilitate deliveryof the compound through skin of a subject, e.g., for delivery of thecompound to the systemic circulation. In one embodiment, the compound isformulated with a penetration enhancer other than DMSO.

In one embodiment, the compound is provided in a drug delivery patch,e.g., a transmucosal or transdermal patch, and can be formulated with apenetration enhancer. The patch generally includes a backing layer,which is impermeable to the compound and other formulation components, amatrix in contact with one side of the backing layer, which matrixprovides for sustained release, which may be controlled release, of thecompound, and an adhesive layer, which is on the same side of thebacking layer as the matrix. The matrix can be selected as is suitablefor the route of administration, and can be, for example, and can be apolymeric or hydrogel matrix.

Depending on the subject and condition being treated and on theadministration route, the subject compounds may be administered indosages of, for example, 0.1 g to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.

A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

For use in the subject methods, the subject compounds may be formulatedwith other pharmaceutically active agents, including otherCFTR-inhibiting agents or agents that block intestinal chloridechannels.

Pharmaceutically acceptable excipients usable with the invention, suchas vehicles, adjuvants, carriers or diluents, are readily available tothe public. Moreover, pharmaceutically acceptable auxiliary substances,such as pH adjusting and buffering agents, tonicity adjusting agents,stabilizers, wetting agents and the like, are readily available to thepublic.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Kits with unit doses of the subject compounds, usually in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the drugs intreating pathological condition of interest. Preferred compounds andunit doses are those described herein above.

Conditions Amenable to Treatment Using the CFTR Inhibitors of theInvention

The CFTR inhibitors disclosed herein are useful in the treatment of aCFTR-mediated condition, i.e., any condition, disorder or disease, orsymptom of such condition, disorder, or disease, that results fromactivity of CFTR, e.g., activity of CFTR in ion transport. Suchconditions, disorders, diseases, or symptoms thereof are amenable totreatment by inhibition of CFTR activity, e.g., inhibition of CFTR iontransport.

In one embodiment, the CFTR inhibitors of the invention are used in thetreatment of conditions associated with aberrantly increased intestinalsecretion, particularly acute aberrantly increased intestinal secretion.CFTR activity has been implicated in intestinal secretion in response tovarious agonists, including cholera toxin (see, e.g., Snyder et al. 1982Bull. World Health Organ. 60:605-613; Chao et al. 1994 EMBO J.13:1065-1072; Kimberg et al. 1971 J. Clin. Invest. 50:1218-1230). ThusCFTR inhibitors of the invention can be administered in an amounteffective to inhibit CFTR ion transport and thus decrease intestinalfluid secretion. In such embodiments, CFTR inhibitors according to theinvention are generally administered by administration to a mucosalsurface of the gastrointestinal tract (e.g., by an enteral route, e.g.,oral, intraintestinal, rectal, and the like) or to a mucosal surface ofthe oral or nasal cavities, or (e.g., intranasal, buccal, sublingual,and the like). In certain embodiments administration of a CFTR inhibitorof the invention that is relatively membrane impermeant (e.g., havingdecreased membrane permeance characteristics (e.g., due to modificationby PEGylation and the like as described above)) is of particularinterest.

Thus, CFTR inhibitors can be used in the treatment of intestinalinflammatory disorders and diarrhea, particularly secretory diarrhea.Secretory diarrhea is the biggest cause of infant death in developingcountries, with about 5 million deaths annually (Gabriel et al., 1994Science 266: 107-109). Several studies, including those using CF mice,indicate that CFTR is the final common pathway for intestinal chlorideion (and thus fluid) secretion in response to various agonists (Snyderet al., 1982, Bull. World Health Organ. 60: 605-613; Chao et al., 1994EMBO. J. 13: 1065-1072; and Kimberg et al., 1971, J. Clin. Invest. 50:1218-1230).

Diarrhea amenable to treatment using the CFTR inhibitors of theinvention can result from exposure to a variety of pathogens or agentsincluding, without limitation, cholera toxin (Vibrio cholera), E. coli(particularly enterotoxigenic (ETEC)), Shigella, Salmonella,Campylobacter, Clostridium difficile, parasites (e.g., Giardia,Entamoeba histolytica, Cryptosporidiosis, Cyclospora), diarrheal viruses(e.g., rotavirus), food poisoning, or toxin exposure that results inincreased intestinal secretion mediated by CFTR.

Other diarrheas include diarrhea associated with AIDS (e.g.,AIDS-related diarrhea), diarrheas caused by anti-AIDS medications suchas protease inhibitors, and inflammatory gastrointestinal disorders,such as ulcerative colitis, inflammatory bowel disease (IBD), Crohn'sdisease, and the like. It has been reported that intestinal inflammationmodulates the expression of three major mediators of intestinal salttransport and may contribute to diarrhea in ulcerative colitis both byincreasing transepithelial Cl⁻ secretion and by inhibiting theepithelial NaCl absorption (see, e.g., Lohi et al., 2002, Am. J.Physiol. Gastrointest. Liver Physiol. 283(3):G567-75).

CFTR inhibitors of the invention can also be used in treatment ofconditions such as polycystic kidney disease, and find further use asmale infertility drugs, by inhibition of CFTR activity in the testis.

CFTR inhibitors of the invention can be further screened in largeranimal models (e.g., the rabbit model described in Spira et al., 1981,Infect. Immun. 32:739-747.). In addition, analysis of stool output usinglive Vibrio cholerae can also be examined to further characterize theCFTR inhibitors of the invention.

Non-Human Animal Models and Human Tissue Models of CFTR-Deficiencies

The CFTR inhibitors of the invention can also be used to generatenon-human animal models of disease, where the disease is associated withdecreased CFTR function (e.g., decreased ion transport). There isincreasing evidence that defective fluid and macromolecular secretion byairway submucosal glands leads to impaired mucociliary and bacterialclearance in CFTR-deficient subjects, particularly in those affectedwith cystic fibrosis (CF); however, functional studies in human airwayglands have been restricted to severely diseased airways obtained at thetime of lung transplantation (Jayaraman et al. 2001 Proc. Natl. Acad.Sci. USA 98:8119-8123). Acute CFTR inhibition permits determination ofthe role of CFTR in water, salt and macromolecule secretion bysubmucosal glands. High-affinity CFTR inhibitors permit thepharmacological creation of non-human animal models that mimicCFTR-deficiency in humans, e.g., mimics the human CF phenotype. Inparticular, large animal models of CFTR deficiency (e.g., CF) findparticular use in elucidating the pathophysiology of initiation andprogression of airway disease in CF, and in evaluating the efficacy ofCF therapies, e.g., screening candidate agents for treatment ofCFTR-deficiencies or symptoms thereof.

Inhibition of CFTR ion transport can be manifested in airway andpancreatic disorders, as well as infertility in males. For example,inhibition of CFTR channels in the lungs and airways influences airwaysurface fluids leading to accumulation of mucus, which in turn plugsairways and collects heavily on the lung walls, providing a primeenvironment for infection to occur, which in turn can lead to chroniclung disease. This same phenomenon occurs in the pancreas, where theaccumulated mucus disrupts the exocrine function of the pancreas andprevents essential food-processing enzymes from reaching the intestines.

Such non-human animal models can be generated by administration of anamount of a CFTR inhibitor effective to decrease CFTR activity in iontransport. Of particular interest is the use of the CFTR inhibitors ofthe invention to induce the cystic fibrosis (CF) phenotype in anon-human animal. Administration of an amount of a CFTR inhibitoreffective to inhibit CFTR in, for example, lung effectively mimics theCFTR defect found in CF. Routes of delivery for CFTR inhibitor arediscussed in detail above. Depending on the non-human animal used, thesubject compounds may be administered in dosages of, for example, 50 to500 μg/kg body weight one to three times a day by an intraperitoneal,subcutaneous, or other route to generate the non-human animal models.Oral dosages may be up to about ten times the intraperitoneal orsubcutaneous dose.

Non-human animal models of CFTR-associated disease can be used as modelsof any appropriate condition associated with decreased CFTR activity.Such conditions include those that are associated with CFTR mutations,which mutations result in abnormalities in epithelial ion and watertransport. These abnormalities can in turn be associated withderangements in airway mucociliary clearance, as well as in othermucosal epithelia and ductal epithelia. Conditions that can bepharmacologically modeled by inducing a CFTR-deficient phenotype in anon-human animal include, without limitation, cystic fibrosis (includingatypical CF), idiopathic chronic pancreatitis, vas deferens defects,mild pulmonary disease, asthma, and the like. For a review of disordersassociated with impaired CFTR function, see, e.g., Noone et al. RespirRes 2 328-332 (2001). CFTR inhibitor-generated non-human animal modelscan also serve as models of microbial infection (e.g., bacterial, viral,or fungal infection, particularly respiratory infections) in aCFTR-deficient subject. In one embodiment of particular interest, theCFTR inhibitors of the invention are used to pharmacologically inducethe cystic fibrosis (CF) phenotype.

Animals suitable for use in the production of the animal models of theinvention include any animal, particularly a mammal, e.g., non-humanprimates (e.g., monkey, chimpanzee, gorilla, and the like), rodents(e.g., rats, mice, gerbils, hamsters, ferrets, and the like),lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline,and the like. Large animals are of particular interest.

The CFTR inhibitors can also be contacted with isolated human tissue tocreate ex vivo models of disease. Such tissue is contacted with anamount of a CFTR inhibitor effective to decrease CFTR activity in thetissue, which may be for as little as 15 minutes, or as much as twohours or more. Human tissues of interest include, without limitation,lung (including trachea and airways), liver, pancreas, testis, and thelike. Physiological, biochemical, genomic or other studies can becarried out on the inhibitor-treated tissue to identify noveltherapeutic target molecules that are important in the pathophysiologyof a disease. For example, isolated tissue from humans without CF can beexposed to inhibitor sufficient to induce the CF phenotype and suchstudies can be carried out to identify novel therapeutic targetmolecules that are important in the pathophysiology of CF.

Synthesis Of The Compounds Of The Invention

Compounds of the invention may be prepared according to methods known toone skilled in the art, or by methods similar to the method describedbelow.

It is understood that in the following description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described below the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),tetrahydropyranyl, benzyl, and the like. Suitable protecting groups foramino, amidino and guanidino include t-butoxycarbonyl,benzyloxycarbonyl, and the like. Suitable protecting groups for mercaptoinclude —C(O)—R (where R is alkyl, aryl or aralkyl), p-methoxybenzyl,trityl and the like. Suitable protecting groups for carboxylic acidinclude alkyl, aryl or aralkyl esters.

Protecting groups may be added or removed in accordance with standardtechniques, which are well-known to those skilled in the art and asdescribed herein.

The use of protecting groups is described in detail in Theodora W.Greene, Peter G. M. Wuts, Protective Groups in Organic Synthesis (1999),3rd Ed., Wiley-Interscience. The protecting group may also be a polymerresin such as a Wang resin or a 2-chlorotrityl chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of formula (I), as described above(e.g., in the Overview and in Hydrazide-Containing Compounds andDerivatives), may not possess pharmacological activity as such, they maybe administered to a mammal and thereafter metabolized in the body toform compounds of the invention which are pharmacologically active. Suchderivatives may therefore be described as “prodrugs”. All prodrugs ofcompounds of formula (I) are included within the scope of the invention.

The following Reaction Schemes illustrate methods to make compounds ofthe invention. It is understood that one of ordinary skill in the artwould be able to make the compounds of the invention by similar methodsor by methods known to one skilled in the art. In general, startingcomponents may be obtained from sources such as Aldrich, or synthesizedaccording to sources known to those of ordinary skill in the art (see,e.g., Smith and March, March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, 5th edition (Wiley Interscience, New York)).Moreover, the various substituted groups (e.g., R₁, R₂, R₃, and X, etc.)of the compounds of the invention may be attached to the startingcomponents, intermediate components, and/or final products according tomethods known to those of ordinary skill in the art.

The following Reaction Scheme 1 is directed to the preparation ofcompounds of formula (I), which are compounds of the invention asdescribed above (e.g., in the Overview and in Hydrazide-ContainingCompounds and Derivatives), where R₁, R₂, and R₃ are as described above(e.g., in the Overview and in Hydrazide-Containing Compounds andDerivatives).

In general, compounds of Formula (I) are prepared by first combining anR₁-group containing a terminal amine containing a Y group with diethyloxalate or an X containing compound such as X-substituted ethyliodoacetate, where X is as described above, each at 10 mmol. Theresulting reaction mixture is then stirred overnight at elevatedtemperature. Upon cooling, the solid material is filtered andrecrystallized from hexane to yield compound of formula (A). A solutionof the compound of formula (A) in ethanol is then refluxed with 12 mmolhydrazine hydrate for a period of time of about 10 hours. The solventand excess reagent are then distilled under vacuum. The product is thenrecrystallized from ethanol to yield the compound of formula (B). Thecompound of formula (B) is then combined with a R₂, R₃-group containingcarbonyl group (e.g., a ketone or an aldehyde) in ethanol and thenrefluxed for a period of time of about 3 hours to yield the desiredproduct of Formula (I).

Alternatively, compounds of Formula (I), where and X is an alkyl groupcontaining X₁, wherein X₁ is an alkyl group such as a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(compounds of Formula Ia) can be prepared according to the followingReaction Scheme 2 wherein R₁, R₂, and R₃ are as described above (e.g.,in the Overview and in Hydrazide-Containing Compounds and Derivatives).

In general, compounds of Formula (Ia) are prepared by first combining anR₁-group containing a terminal amine containing an Y group with ethyliodoacetate containing an X₁ group, where X₁ is as described above, eachat 10 mmol with 20 mmol sodium acetate. The resulting reaction mixtureis then stirred at elevated temperature for a period of time of about 3hours. Upon cooling, the solid material is filtered and recrystallizedfrom hexane to yield compound of formula (C). A solution of the compoundof formula (C) in ethanol is then refluxed with 12 mmol hydrazinehydrate for a period of time of about 10 hours. The solvent and excessreagent are then distilled under vacuum. The product is thenrecrystallized from ethanol to yield the compound of formula (D). Thecompound of formula (D) is then combined with a R₂, R₃-group containingcarbonyl group (e.g., a ketone or an aldehyde) in ethanol and thenrefluxed for a period of time of about 3 hours to yield the desiredproduct of Formula (Ia).

The following Reaction Scheme 3 is directed to the preparation ofcompounds of Formula (Ib) wherein X is CH₂, which are compounds of theinvention as described above (e.g., in the Overview and inHydrazide-Containing Compounds and Derivatives), where R₁, R₂, and R₃are as described above (e.g., in the Overview and inHydrazide-Containing Compounds and Derivatives).

In general, compounds of Formula (Ib) are prepared by first combining anR₁-group containing a terminal amine with ethyl iodoacetate each at 10mmol with 20 mmol sodium acetate. The resulting reaction mixture is thenstirred at an elevated temperature for a period of time of about 3hours. Upon cooling, the solid material is filtered and recrystallizedfrom hexane to yield compound of formula (E). A solution of the compoundof formula (E) in ethanol is then refluxed overnight with 12 mmolhydrazine hydrate for a period of time of about 10 hours. The solventand excess reagent are then distilled under vacuum. The product is thenrecrystallized from alcohol to yield the compound of formula (F). Thecompound of formula (F) is then combined with a R₂, R₃-group containingcarbonyl group (e.g., a ketone or an aldehyde) in ethanol and thenrefluxed for a period of time of about 3 hours to yield the desiredproduct of Formula (Ib).

Alternatively, compounds of Formula (I), where X is a carbonyl group(compounds of Formula Ic) can be prepared according to the followingReaction Scheme 4 wherein R₁, R₂, and R₃ are as described above in theOverview.

In general, compounds of Formula (Ic) are prepared by first combining anR₁-group containing a terminal amine containing a Y group with diethyloxalate each at 10 mmol in toluene. The resulting reaction mixture isthen stirred at an elevated temperature for a period of time of about 3hours. Upon cooling, the solid material is filtered and recrystallizedfrom hexane to yield compound of formula (G). A solution of the compoundof formula (G) in ethanol is then refluxed with 12 mmol hydrazinehydrate for a period of time of about 10 hours. The solvent and excessreagent are then distilled under vacuum. The product is thenrecrystallized from ethanol to yield the compound of formula (H). Thecompound of formula (H) is then combined with a R₂, R₃-group containinga carbonyl group in ethanol and then refluxed for a period of time ofabout 3 hours to yield the desired product of Formula (Ic).

Alternatively, compounds of Formula (I), where X is an alkyl groupcontaining Y″ (compounds of Formula Ie) can be prepared according to thefollowing Reaction Schemes 5-8 wherein R₁, R₂, and R₃ are as describedabove (e.g., in the Overview and in Hydrazide-Containing Compounds andDerivatives), and wherein Y″ is independently chosen from an substitutedor unsubstituted alkyl group, such as a substituted or unsubstituted,saturated linear or branched hydrocarbon group or chain (e.g., C₁ to C₈)including, e.g., methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl,dodecyl, octadecyl, amyl, 2-ethylhexyl; an alkyl group carrying polargroups such as hydroxy, sulfo, carboxylate, and a substituted orunsubstituted carboxamide groups (where exemplary groups include, suchas 3-sulfopropyl, 4-sulfobutyl, carboxymethyl, 2-carboxypropyl,2-methoxy-2-oxoethyl, 3-methoxy-3-oxoproplyl); or a linker such as anamide bond or ether linker to provide for attachment of one or more tolarger polar molecules, such as substituted or unsubstituted phenylgroup, a polyoxyalkyl polyether (such as polyethylene glycol (PEG),polypropylene glycol, polyhydroxyethyl glycerol), polyethyleneimines,disaccharides, trisaccharides, polyalkylimines, small amino dextrans andthe like, where Y″ can further include such an attached polarmolecule(s).

In general, in some embodiments, compounds of Formula (Ie) are preparedby first combining an R₁-group containing a terminal amine containing aY group with diethyl bromomalonate each at 10 mmol. The resultingreaction mixture is then stirred at an elevated temperature for a periodof time of about 8 hours. Upon cooling, the solid material is filteredand recrystallized from hexane to yield compound of formula (I). Asolution of the compound of formula (I) in ethanol is then refluxed with12 mmol hydrazine hydrate for a period of time of about 10 hours. Thesolvent and excess reagent are then distilled under vacuum. The productis then recrystallized from ethanol to yield the compound of formula(J). The compound of formula (J) is then combined with a R₂, R₃-groupcontaining a carbonyl group in ethanol and then refluxed for a period oftime of about 3 hours to yield the desired product of Formula (K). Thecompound of formula (K) is then combined with a substituted orunsubstituted phenyl group as described in greater detail above (e.g.,in the Overview and in Hydrazide-Containing Compounds and Derivatives)and refluxed for a period of time. The product is then recrystallizedfrom ethanol to yield the compound of formula (L).

In general, in some embodiments, compounds of Formula (Ie) are preparedby first combining an R₁-group containing a terminal amine containing aY group with diethyl bromomalonate each at 10 mmol. The resultingreaction mixture is then stirred at an elevated temperature for a periodof time of about 8 hours. Upon cooling, the solid material is filteredand recrystallized from hexane to yield compound of formula (I). Asolution of the compound of formula (I) in ethanol is then refluxed with12 mmol hydrazine hydrate for a period of time of about 10 hours. Thesolvent and excess reagent are then distilled under vacuum. The productis then recrystallized from ethanol to yield the compound of formula(J). The compound of formula (J) is then combined with a R₂, R₃-groupcontaining a carbonyl group in ethanol and then refluxed for a period oftime of about 3 hours to yield the desired product of Formula (K). Thecompound of formula (K) is then combined with a thiocyanate substitutedphenyl group as described in greater detail above (e.g., in the Overviewand in Hydrazide-Containing Compounds and Derivatives) and refluxed fora period of time. The product is then recrystallized from ethanol toyield the compound of formula (M).

In general, in some embodiments, compounds of Formula (Ie) are preparedby first combining a thiocyanate containing a phenyl group withAmino-PEG in DMF and stirred at an elevated temperature for a period oftime of about 24 hours. The DMF is then evaporated in vacuo, and theresidue is dissolved in minimal quantity EtOAc and added to a stirredsolution of Et₂O. The resulting precipitate is then filtered and washedin Et₂O to give the PEG-containing compound. The PEG-containing compoundis then combined with the compound of formula (K) and refluxed for aperiod of time. The product is then recrystallized from ethanol to yieldthe compound of formula (N).

In general, in some embodiments, compounds of Formula (Ie) are preparedby first combining a thiocyanate containing a phenyl group withAmino-PEG in DMF and stirred at an elevated temperature for a period oftime of about 24 hours. The DMF is then evaporated in vacuo, and theresidue is dissolved in minimal quantity EtOAc and added to a stirredsolution of Et₂O. The resulting precipitate is then filtered and washedin Et₂O to give the PEG-containing compound. The PEG-containing compoundis then combined with the compound of formula (K) and refluxed for aperiod of time. The product is then recrystallized from ethanol to yieldthe compound of formula (O).

In general, in some embodiments, compounds of Formula (Ie) are preparedby first combining an R₁-group containing a terminal amine containing aY group with diethyl bromobuterolacetone or bromobuterolactone each at10 mmol. The resulting reaction mixture is then stirred at an elevatedtemperature for a period of time of about 8 hours. Upon cooling, thesolid material is filtered and recrystallized from hexane to yieldcompound of formula (P). A solution of the compound of formula (P) inethanol is then refluxed with 12 mmol hydrazine hydrate for a period oftime of about 10 hours. The solvent and excess reagent are thendistilled under vacuum. The product is then recrystallized from ethanolto yield the compound of formula (Q). The compound of formula (Q) (10mM) is then combined with 20 mM (BOC)₂O in 10 mL of THF and heated underreflux conditions for a period of time of about 5 hours. The solvent isthen removed, and the residue is purified by column chromatography onsilica gel and eluted with dichloromethane to give the compound offormula (R). The compound of formula (R) (1 mmol) is then combined withTsCl (1 mmol) in pyridine (5 ml) in three portion a period of time ofabout 30 min apart form one another. The reaction mixture is thenstirred at a low temperature of about −15° C. for a period of time ofabout 8 hours. The reaction mixture is then allowed to warm to roomtemperature, diluted with 1N HCl, and then extracted three times withEtOAc. The combined organic extract is then washed with brine, driedwith NaSO4 and evaporated to dryness to give the compound of formula(S). The compound of formula (S) is then combined with amino-PEG, suchas 2-aminoethoxyethanol, in DMF and stirred at an elevated temperaturefor a period of time of about 24 hours. The DMF is then evaporated invacuo, and the residue is dissolved in minimal quantity EtOAc and addedto a stirred solution of Et₂O. The resulting precipitate is thenfiltered and washed in Et₂O to give the compound of formula (T). Thecompound of formula (T) is then dissolved in minimal amount oftrifluoroacetic acid:Ch₂Cl₂ (1:1) and stirred at room temperature for aperiod of time of about 30 minutes. The reaction mixture is the dilutedwith saturated aqueous NaHCO₃ and extracted with CH₂Cl₂. The combinedorganic layer is then washed successively with water and brine, driedand concentrated in vacuo to yield the compound of formula (U). Thecompound of formula (U) is then combined with a R₂, R₃-group containinga carbonyl group in ethanol and then refluxed for a period of time ofabout 3 hours to yield the desired product of Formula (V).

Structures were confirmed by ¹H-NMR and Mass spectrometry. Puritywas >98% as judged by thin layer chromatography and HPLC.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Method and Materials

The following materials and methods were used in the examples thatfollow.

High-Throughput Screening for Identification of CFTR Inhibitors

Screening was performed using an integrated system (Beckman) consistingof a 3-meter robotic arm, CO₂ incubator, plate washer, liquid handlingwork station, barcode reader, delidding station, plate sealer and twofluorescence plate readers (Optima, BMG Lab Technologies), each equippedwith two syringe pumps and HQ500/20X (500±10 nm) excitation andHQ535/30M (535±15 nm) emission filters (Chroma).

One hundred thousand small molecules (most 350-550 daltons) wereselected for screening from commercial sources (ChemBridge and ChemDiv,both of San Diego, Calif.) using algorithms designed to maximizechemical diversity and drug-like properties. The compounds were obtainedas a dried powder and solutions were made in DMSO just before testing,stored frozen as 2.5 mM stock solutions for further use.

Fisher Rat Thyroid (FRT) cells stably expressing wildtype human CFTR andYFP-H148Q were cultured on 96-well black wall plates as describedpreviously (Ma et al., J. Biol. Chem., 277:37235-37241, 2002). Forscreening, cells in 96-well plates were washed three times and then CFTRhalide conductance was activated by incubation for 15 minutes with anactivating cocktail containing 10 μM forskolin, 20 μM apigenin and 100μM isobutylmethyl-xanthine (IBMX). Test compounds (final 25 μM) wereadded 5 minutes prior to assay of iodide influx in which cells wereexposed to a 100 mM inwardly-directed iodide gradient. YFP fluorescencewas recorded for 2 seconds prior to and 12 seconds after creation of theiodide gradient. Initial rates of iodide influx were computed from thetime course of decreasing fluorescence after the iodide gradient (Yanget al., J. Biol. Chem., 35079-35085, 2003).

Short-Circuit Current Measurements

FRT, T84 colon epithelial cells and human airway epithelial cells werecultured on Snapwell filters with 1 cm² surface area (Corning-Costar) toresistances>1,000 Ω·cm² as described previously (Ma et al., J. Biol.Chem., 277:37235-37241, 2002). Filters were mounted in an EasymountChamber System (Physiologic Instruments, San Diego). For apical Cl⁻current measurements on FRT cells, the basolateral hemichamber wasfilled with buffer containing (in mM): 130 NaCl, 2.7 KCl, 1.5 KH₂PO₄, 1CaCl₂, 0.5 MgCl₂, 10 Na-HEPES, 10 glucose (pH 7.3). The basolateralmembrane was permeabilized with amphotericin B (250 μg/ml) just prior tomeasurements. In the apical solution 65 mM NaCl was replaced by sodiumgluconate, and CaCl₂ was increased to 2 mM. For short-circuit currentmeasurements in (non-permeabilized) T84 and human airway cells, bothhemichambers contained Kreb's solution (in mM): 120 NaCl, 25 NaHCO₃, 3.3KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂ and 10 glucose (pH 7.3).Solutions were bubbled with 95% O₂ and 5% CO₂ and maintained at 37° C.For studies in mouse intestine, ileal segments were isolated, washedwith ice-cold Kreb's buffer, opened longitudinally through themesenteric border, and mounted in a micro-Ussing chamber (0.7 cm²aperture area, World Precision Instruments). Hemichambers were filledwith Kreb's solutions containing 10 μM indomethacin. ApicalCl⁻/short-circuit current were recorded using a DVC-1000 voltage-clamp(World Precision Instruments) with Ag/AgCl electrodes and 1 M KCl agarbridges.

Patch-Clamp Analysis

Patch-clamp experiments were carried out at room temperature on FRTcells stably expressing wildtype CFTR. Cell-attached and whole-cellconfigurations were used (Hamill et al., Pflugers Arch. 391:85-100,1981). The cell membrane was clamped at specified voltages using anEPC-7 patch-clamp amplifier (List Medical). Data were filtered at 500 Hzand digitized at 2000 Hz. For whole-cell experiments the pipettesolution contained (in mM): 120 CsCl, 10 TEA-Cl, 0.5 EGTA, 1 MgCl₂, 40mannitol, 10 Cs-HEPES and 3 mM MgATP (pH 7.3). For cell attachedexperiments EGTA was replaced with 1 mM CaCl₂. The bath solution forwhole-cell experiments contained (in mM): 150 NaCl, 1 CaCl₂, 1 MgCl₂, 10glucose, 10 mannitol, 10 Na-TES (pH 7.4). In cell-attached experimentsthe bath solution contained (in mM): 130 KCl, 2 NaCl, 2 CaCl₂, 2 MgCl₂,10 glucose, 20 mannitol, and 10 K-Hepes (pH 7.3). Inhibitors wereapplied by extracellular perfusion. CFTR channel activity incell-attached patches was analyzed as described previously (Taddei etal., FEBS Lett. 558:52-56, 2004).

Nasal Potential Difference Measurements in Mice

Following anesthesia with intraperitoneal ketamine (90-120 mg/kg) andxylazine (5-10 mg/kg) the airway was protected by orotracheal intubationwith a 21-gauge angiocatheter as described. A PE-10 cannula pulled to atip diameter of 0.3 mm was inserted into one nostril 5 mm distal to theanterior nares and connected though a 1M KCl agar bridge to a Ag/AgClelectrode and high-impedance digital voltmeter (IsoMillivolt Meter,World Precision Instruments). The nasal cannula was perfused at 50μL/min using dual microperfusion pumps serially with PBS, low chloridePBS (chloride replaced by gluconate), low chloride PBS containingforskolin (10 μM) without and then with GlyH-101 (10 μM), and then PBS.In some studies GlyH-101 (10 μM) or4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) (100 μM) waspresent in all solutions. The reference electrode was a PBS-filled21-gauge needle inserted in the subcutaneous tissue in the abdomen andconnected to a second Ag/AgCl electrode by a 1M KCl agar bridge.

Intestinal Fluid Secretion Measurements

Mice (CD1 strain, 25-35 g) were deprived of food for 24 hr andanaesthetized with intraperinoneal ketamine (40 mg/kg) and xylazine (8mg/kg). Body temperature was maintained at 36-38° C. using a heatingpad. Following a small abdominal incision 3 closed ileal loops (length20-30 mm) proximal to the cecum were isolated by sutures. Loops wereinjected with 100 μl of PBS or PBS containing cholera toxin (1 μg)without or with GlyH-101 (2.5 μg). The abdominal incision was closedwith suture and mice were allowed to recover from anesthesia. At 4hours, the mice were anesthestized, intestinal loops were removed, andloop length and weight were measured to quantify net fluid secretion.

Cholera Models

For closed loop studies, mice (CD1 strain, 28-34 g) were deprived offood for 24 hours and then anaesthetized with intraperinoneal ketamine(40 mg/kg) and xylazine (8 mg/kg). Body temperature was maintained at36-38° C. using a heating pad. Following a small abdominal incisionthree closed mid-jejunal loops (length 15-20 mm) were isolated bysutures. Loops were injected with 100 μl of PBS or PBS containingcholera toxin (1 μg) without or with test compounds. The abdominalincision was closed with suture and mice were allowed to recover fromanesthesia. At 4 hours the mice were anesthestized, intestinal loopswere removed, and loop length and weight were measured to quantify netfluid secretion. Mice were sacrificed by an overdose of ketamine andxylazine. All protocols were approved by the UCSF Committee on AnimalResearch.

Intestinal Absorption Studies

Absorption studies were performed using mid-jejunal loops created asdescribed above. Loops were injected separately with MalH-1, MalH-2,MalH-3, MalH-(PEG)_(n), and GlyH-(PEG)_(n) containing 10-20 μg of testcompounds together with 5 μg FITC-dextran (40 kDa). After 2 hours loopfluid was withdrawn and optical absorbance of test compound and FITCwere measured (OD₃₄₂/OD_(494nm)). Percentage intestinal absorption wascomputed assuming zero absorption of FITC-dextran.

Synthesis of Compounds

The synthesis of compounds of the invention are exemplified with but notlimited to the following examples. All synthesized compounds were >98%pure (TLC/HPLC) and were confirmed by mass and ¹H nmr spectrometry.

Synthesis ofN-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide (GlyH-101) and related glycine hydrazides (GlyH-102-109,114-127)

A mixture of 2-napthylamine (compound I, FIG. 3B) (1.43 g, 10 mmol),ethyl iodoacetate (2.14 g, 10 mmol), and sodium acetate (1.64 g, 20mmol, dissolved in 2 ml of water) was stirred at 90° C. for 3 hours. Thesolid material obtained upon cooling was filtered and recrystallizedfrom hexane to yield 1.5 g ethyl N-(2-naphthalenyl)glycinate (compoundII, FIG. 3B) (yield, 65%, mp 83-84° C.) (Ramamurthy and Bhatt, J. Med.Chem. 32:2421-2426, 1989). A solution of above product (2.29 g, 10 mmol)in ethanol (10 ml) was refluxed with hydrazine hydrate (0.6 g, 12 mmol)for 10 hours. Solvent and excess reagent were distilled under vacuum.The product was recrystallized from ethanol to yield 1.8 g ofN-(2-naphthalenyl) glycine hydrazide (compound III, FIG. 3B) (yield 82%,mp 147-148° C.). A mixture of compound III (2.15 g, 10 mmol) and3,5-dibromo-2,4-dihydroxybenzaldehyde (3 g, 10 mmol) in ethanol (5 ml)was refluxed for 3 hours. The hydrazone that crystallized upon coolingwas filtered, washed with ethanol, and recrystallized from ethanol togive 3.8 g (78%) of GlyH-101. Melting point (mp)>300° C., ms (ES⁻): M/Z492 (M⁻); ¹H nmr (DMSO-d₆): δ 4.1 (s, 2H, CH₂), 6.5-7.5 (m, 9H,aromatic, NH), 8.5 (s, 1H, CH═N), 10.4 (s, 1H, NH—CO), 11.9 (s, 1H, OH),12.7 (s, 1H, OH). Compounds GlyH-102-109, GlyH-114-127 and AceH401-404were synthesized similarly by condensing appropriate hydrazides withsubstituted benzaldehydes.

Synthesis ofN-(6-quinolinyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide (GlyH-126) and related quinolinyl-glycine hydrazides

To a stirred solution of 6-aminoquinoline (compound IV, FIG. 3B) (0.72g, 5 mmol) in acetonitrile (20 ml) was added 33% aqueous glyoxylic acid(1.85 g, 20 mmole) solution. A solution of NaBH₃CN (0.64 g, 10.2 mmol)in acetonitrile (20 ml) was then added at 3° C. over 20 minutes and thereaction mixture was warmed to room temperature and stirred for 48hours. Acetonitrile was evaporated under vacuum, water (20 ml) was addedto the residue, the solution was alkalinized to pH 9.5, and unreactedamine was extracted with ether. Concentrated HCl (25 ml) was added tothe aqueous solution and the mixture was stirred at 25° C. for 1 hour.Solvent was evaporated under vacuum. The resultant residue ofN-(6-quinolinyl)glycine was dissolved in dry ethanol (50 ml) saturatedwith dry HCl, stirred overnight and then refluxed for 3 hours. Ethanolwas evaporated, the ester hydrochloride was suspended in dry ether, andammonia gas was bubbled. The ammonium chloride was filtered and etherwas removed by evaporation to give ethyl N-(6-quinolinyl)glycinate (0.5g, 87%, mp 122-123° C.). N-(6-quinolinyl)glycine hydrazide (compound VI,FIG. 3B), synthesized by hydrazinolysis of the above ester, was reactedwith 3,5-dibromo-2,4-dihydroxybenzaldehyde to give GlyH-126. Similarprocedures were used for synthesis of GlyH-127.

Synthesis of Oxamic Hydrazides (OxaH-110-113)

The oxamic hydrazides were synthesized by heating a mixture of2-napthaleneamine with diethyl oxalate in toluene. The resultantN-substituted oxamic acid ethyl ester was treated with hydrazine hydratefollowed by condensation with substituted benzaldehydes to yieldcompounds OxaH-110-113.

Synthesis of 3,5-dibromo-4-hydroxy-[2-(2-napthalenamine)aceto]benzoicacid hydrazide (GlyH-202) and related GlyH-201 and Oxa-203-204

N-(2-naphthalenyl)glycine hydrazide (compound III, FIG. 3B) (2.15 g, 10mmole) was reacted with 3,5-dibromo-4-hydroxybenzoyl chloride (3.14 g,10 mmole) (Gilbert et. al., Eur. J. Med. Chem., 17:581-588, 1982) inpyridine (10 ml) for 5 hours. Pyridine was removed and the residue wasdiluted with water. The product was recrystallized from ethanol to yielda gray powder 3.8 g (77%), mp>300° C. Compounds GlyH-201 and Oxa-203-204were synthesized by similar procedure.

Synthesis ofN-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methyl]glycinehydrazide (GlyH-301) and related glycine hydrazides (GlyH-302OxaH-303-304)

A mixture of GlyH-101 (1.5 g, 3 mmole), hydrazine hydrate (0.15 ml, 3mmol) and Pd/C catalyst (0.1 g, 10% Pd) in 5 ml of dimethylformamide wasrefluxed for 6-8 hours (Verma et al., Arch. Pharm. 317:890-894, 1984).The reaction mixture was filtered, diluted with cold water, andextracted with diethyl ether. GlyH-301 was crystallized from ether toyield 0.9 g (60%), mp 258-260° C. Compounds GlyH-302 and OxaH-303-304were prepared similarly.

Synthesis of Analogs

The synthesis of analog of the compounds of the invention areexemplified with but not limited to the following examples. Allsynthesized compounds were >98% pure (TLC/HPLC) and were confirmed bymass and ¹H nmr spectrometry. ¹H NMR spectra were obtained in CDCl₃ orDMSO-d₆ using a 400 MHz Varian Spectrometer referenced to CDCl₃ or DMSO.Mass spectrometry was done using a Waters LCMS system (Alliance HT2790+ZQ, HPLC: Waters model 2960, Milford, Mass.). Flash chromatographywas performed using EM silica gel (230-400 mesh), and thin layerchromatography was done on Merk silica gel 60 F254 plates.

Synthesis of Diethyl-(2-naphthalenylamino)-propanedioate (compound 2FIG. 9)

A mixture of 2-naphthylamine (compound 1, FIG. 9) (10 mmol), diethylbromomaloante (10 mmol), and sodium acetate (1.64 g, 20 mmol, dissolvedin 4 ml of water) was stirred at 90° C. for 8 hours. The black solidmaterial obtained upon cooling was filtered and recrystallized fromhexane to yield 2.5 g of 2 (yield 84%); mp, 189-190° C.; ms (ES⁺): M/Z302 (M+1)⁺; ¹H nmr (DMSO-d₆): δ 1.17 (t, 6H, 7.33 Hz), 4.17 (q, 4H,7.33), 5.10 (d, 1H, 8.79 Hz), 6.54 (d, 1H, 8.79 Hz), 6.75 (d, 1H, 2.20Hz), 7.13 (t, 1H, 7.32 Hz), 7.19 (dd, 1H, 2.19, 8.79 Hz), 7.28 (t, 1H,8.06 Hz), 7.51 (d, 1H, 8.42 Hz), 7.61 (t, 2H, 8.79 Hz).

Synthesis of (2-naphthalenylamino)-propanedioic acid dihydrazide(compound 3 FIG. 9)

A solution of compound 2 (FIG. 9) (10 mmol) in ethanol (10 ml) wasrefluxed with hydrazine hydrate (12 mmol) for 10 hours. Solvent andexcess reagent were distilled under vacuum. The product wasrecrystallized from ethanol to give 2.5 g of compound 3 (92%); mp268-270° C.; ms (ES⁺): M/Z 274 (M+1)⁺; ¹H nmr (DMSO-d₆): δ 4.29 (d, 4H,4.03), 4.56 (d, 1H, 8.79 Hz), 6.03 (d, 1H, 8.79 Hz), 6.62 (d, 1H, 1.46Hz), 7.09 (m, 2H), 7.28 (t, 1H, 8.05 Hz), 7.50 (d, 1H, 8.06 Hz), 7.61(m, 2H), 9.22 (s, 2H).

Synthesis of2-naphthalenylamino-bis[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propanedioicacid dihydrazide (MalH-1)

A mixture of compound 3 (FIG. 9) (10 mmol) and3,5-dibromo-2,4-dihydroxybenzaldehyde (20 mmol) in ethanol (5 ml) wasrefluxed for 3 hours. The hydrazone that crystallized upon cooling wasfiltered, washed with ethanol, and purified by column chromatography(silica gel EtOAc:hexane 2:3) to give 3.2 g of compound 4 (58%) as anoff-white solid; mp 246-248° C.; ms (ES⁺): M/Z 830 (M+1)⁺; ¹H nmr(DMSO-d₆): δ 4.91, 5.48 (d, 1H, 7.69, 9.15 Hz), 6.62 (d, 1H, 7.32 Hz),6.73, 6.84 (s, 1H), 7.13-7.32 (m, 3H), 7.57 (d, 1H, 8.06 Hz), 7.61-7.70(m, 3H), 7.80, 7.90 (s, 1H), 8.15, 8.37 (s, 2H), 10.10-10.40 (broad s,2H), 11.72, 11.90 (s, 2H), 12.22, 12.53 (s, 2H).

Synthesis of2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][(2,4-disodium-disulfophenyl)methylene]propanedioicacid dihydrazide (MalH-2)

A mixture of dihydrazide 4 (FIG. 9) (5 mmol) and2,4-disodium-disulfobenzaldehyde (5 mmol) in DMF (5 ml) was refluxed for4 hours. The reaction mixture, upon cooling, was added dropwise to astirred solution of EtOAc:EtOH (1:1), filtered, washed with ethanol, andfurther purified by column chromatography (silica gel EtOAc:hexane 2:3)to give 2.3 g of compound MalH-2 (58%) as an off-white solid; mp>300°C.; ms (ES⁺): M/Z 800 (M+1)⁺; ¹H nmr (DMSO-d₆): δ 4.95, 5.44 (d, 1H,7.63, 9.16 Hz), 6.64 (d, 1H, 7.31 Hz), 6.70, 6.81 (s, 1H), 7.12-7.44 (m,4H), 7.59 (d, 1H, 8.00 Hz), 7.64-7.76 (m, 4H), 7.80, 7.90 (s, 1H), 8.25,8.37 (s, 2H), 10.36 (broad s, 1H), 11.62, 11.82 (s, 1H), 12.11, 12.43(s, 2H).

2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-(4-sodium-sulfophenyl)-thioureido]propanedioicacid dihydrazide (MalH-3) and2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-[4-(3-(PEG)_(n)-thioureido)phenyl)-thioureido]propanedioicacid dihydrazide (MalH-(PEG)_(n)) were synthesized following similarreaction conditions used for MalH-2 except that4-sodium-sulfophenylisothiocyanate and compound 6 (FIG. 10) were usedrespectively, in place of 2,4-disodium-disulfobenzaldehyde.

MalH-3: mp>300° C.; ms (ES⁻): M/Z 765 (M−1)⁺; ¹H nmr (DMSO-d₆): δ 4.90,5.31 (d, 1H, 7.61, 9.12 Hz), 6.54 (d, 1H, 7.31 Hz), 6.70, 6.81 (s, 1H),7.12-7.44 (m, 4H), 7.59 (d, 1H, 8.00 Hz), 7.64-7.76 (m, 4H), 7.90 (d,2H), 8.25, 8.37 (s, 1H), 9.88 (s, 1H) 10.05 (s, 1H, CSNH), 10.36 (s, 1H,OH), 11.11, 11.43 (s, 2H, CONH), 11.62, 11.82 (s, 1H, OH).

MalH-(PEG)₁: mp>300° C.; ms (ES⁺): M/Z 849 (M+1)⁺; ¹H nmr (DMSO-d₆): δ3.70-4.37 (m, 8H), 4.81, 5.01 (d, 1H, 7.51, 9.13 Hz), 5.27 (s, 1H), 6.60(d, 1H, 7.31 Hz), 6.75 (s, 1H), 7.19-7.38 (m, 4H), 7.59 (d, 2H, 8.00Hz), 7.64-7.76 (m, 3H), 7.90 (d, 2H, 8.00 Hz), 8.21, 8.30 (s, 1H), 9.76(s, 2H) 9.83 (s, 1H), 10.01 (s, 1H), 10.36 (s, 1H), 11.20, 11.51 (s,2H), 11.54, 11.62 (s, 1H).

Synthesis of 2-[3-(4-isothiocyanato-phenyl)-thioureido]ethyl-(PEG)₁(compound 6a, FIG. 10)

To a solution of 1,4-phenylene diisothiocyanate (1 mmol, 2 mL DMF) wasadded 2-aminoethoxyethanol (0.3 mmol, 2 mL DMF) over 30 minutes. Afterstirring for additional 30 minutes, the DMF was distilled off andproduct was purified by column chromatography on silica gel using assolvent n-hexane:AcOEt (1:1). Fractions were evaporated to give 58 mg ofcompound 2 in (65%); ms (ES⁺): M/Z 298 (M+1)⁺; ¹H nmr (DMSO-d₆): δ 2.84(t, 2H, 6.46 Hz), 2.95 (t, 2H, 6.31 Hz), 3.12 (t, 2H, 6.38 Hz), 3.58 (q,2H, 5.98 Hz), 5.63 (s, 1H), 7.15 (d, 2H, 8.62 Hz), 7.44 (d, 2H, 8.62Hz), 7.97 (s, 2H, NH). Similarly, compound 6b was synthesized usingappropriate amino-PEG; yield, 58%; ms (ES): M/Z 736 (+/−44, 88, 132,176) (M+1); ¹H nmr (DMSO-d₆): δ 3.24 (s, 3H), 3.31-3.82 (m), 7.21 (d,2H, 8.60 Hz), 7.47 (d, 2H, 8.60 Hz), 7.92 (s, 2H).

Synthesis of 2-(2-naphthalenylamino)-4-hydroxy-butyric acid hydrazide(compound 7 FIG. 11)

This compound was synthesized following similar reaction conditions usedfor compounds 2 and 3.89%; mp 258-260° C.; ms (ES⁺): M/Z 260 (M+1)⁺; ¹Hnmr (DMSO-d₆): δ 1.79 (m, 2H) 3.46 (q, 2H) 3.98 (s, 1H), 4.17 (d, 2H)4.52 (t, 1H), 5.94-5.96 (s, 1H), 6.68 (s, 1H), 6.98 (dd, 1H), 7.05 (t,1H), 7.24 (t, 1H), 7.46 (d, 1H), 7.52-7.60 (m, 2H) 9.17 (s, 1H).

Synthesis of [2-(2-naphthalenylamino)-4-hydroxy]butyricacid-2-[(1,1-dimethylethoxy)carbonyl]hydrazide (compound 8 FIG. 11)

To a solution of hydrazide 7 (10 mM) in THF (10 ml) was added (BOC)₂O(20 mM) and heated under reflux for 5 hours. The solvent was removed,and the residue was purified by column chromatography on silica gel.Elution with dichloromethane gave 3.1 g of compound 8 (86%) as a whitesolid; mp 235-237° C.; ms (ES⁺): M/Z 360 (M+1)⁺; ¹H nmr (DMSO-d₆): δ1.33 (s, 9H), 1.92 (m, 2H), 3.52 (q, 2H), 4.01 (q, 1H), 4.52 (t, 1H),6.00 (d, 1H), 6.70 (s, 1H), 6.97 (dd, 1H), 7.06 (t, 1H), 7.25 (t, 1H),7.45 (d, 1H), 7.52-7.59 (m, 2H), 8.73 (s, 1H), 9.77 (s, 1H).

Synthesis of [2-(2-naphthalenylamino)-4-(p-tosyl)]butyricacid-2-[(1,1-dimethylethoxy)carbonyl]hydrazide (compound 9 FIG. 11)

To a solution of hydrazide 7 (1 mmol) in pyridine (5 ml) was addedp-TsCl (1 mmol) in three portions 30 min apart (−15° C.). The reactionmixture was stirred for 8 hours at −15° C., allowed to warm to roomtemperature, diluted with 1N HCl, and extracted three times with EtOAc.The combined organic extract was washed with brine, dried with Na₂SO₄and evaporated to dryness to give 374 mg of compound 9 (73%) as a paleyellow oil, used without further purification for next step; ms (ES⁺):M/Z 514 (M+1)⁺.

Synthesis of [2-(2-naphthalenylamino)-4-(PEG-amino)]butyricacid-2-[(1,1-dimethylethoxy)carbonyl]hydrazide (compound 10 FIG. 11)

A solution of 2-aminoethoxyethanol (1 mM) and compound 9 (1 mM) in DMF(2 ml) was stirred at 80° C. for 24 hours. The DMF was evaporated invacuo, and the residue was dissolved in minimum quantity of EtOAc andadded to a stirred solution of Et₂O. The white powder-like precipitatewas filtered and washed with Et₂O to give 170 mg of compound 9 (38%) asa yellow sticky mass; ms (ES⁺): M/Z 447 (M+1)⁺; ¹H nmr (DMSO-d₆): δ 1.35(s, 9H), 1.71 (m, 2H) 3.40-3.51 (m, 4H), 3.57 (t, 2H), 3.68-3.79 (m, 5H,CH2), 3.93 (s, 1H), 4.52 (t, 1H), 6.04, 6.16 (s, 1H), 6.67 (s, 1H), 6.93(dd, 1H), 7.03 (t, 1H), 7.32 (t, 1H), 7.45 (d, 1H), 7.50-7.62 (m, 2H),9.27 (s, 1H), 9.89 (s, 1H).

Synthesis of [2-(2-naphthalenylamino)-4-(PEG-amino)]butyric acidhydrazide (compound 11FIG. 11)

Hydrazide 10 (1 mM) was dissolved in a minimal amount of trifluoroaceticacid:CH₂Cl₂ (1:1) and stirred at room temperature for 30 minutes. Thereaction mixture was diluted with saturated aqueous NaHCO₃ and extractedwith CH₂Cl₂. The combined organic layer was washed successively withwater and brine, dried (Na₂SO₄), and concentrated in vacuo to yield 253mg of compound 11 (73%) as yellow semisolid; ms (ES⁺): M/Z 347 (M+1)⁺;¹H nmr (DMSO-d₆): ¹H nmr (DMSO-d₆): ¹H nmr (DMSO-d₆): δ 1.71 (m, 2H)3.40-3.51 (m, 4H), 3.57 (t, 2H), 3.68-3.79 (m, 5H, CH2), 3.93 (s, 1H),4.26 (d, 2H) 4.52 (t, 1H), 6.02, 6.21 (s, 1H), 6.71 (s, 1H), 6.85 (dd,1H), 7.10 (t, 1H), 7.34 (t, 1H), 7.51 (d, 1H), 7.53-7.76 (m, 2H), 9.27(s, 1H).

Synthesis of [2-(2-naphthalenylamino)-4-(PEG-amino)]butyricacid-2-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]hydrazide (compound12 FIG. 11)

A mixture of compound 11 (1 mmol) and3,5-dibromo-2,4-dihydroxybenzaldehyde (1 mmol) in ethanol (2 ml) wasrefluxed for 3 hours. The reaction mixture was concentrated and added toa stirred solution of Et₂O, and the precipitated hydrazone was filteredand washed with Et₂O to yield 362 mg of compound 12 (58%); ms (ES⁺): M/Z625 (M+1)⁺; ¹H nmr (DMSO-d₆): ¹H nmr (DMSO-d₆): δ 1.75 (m, 2H) 3.43-3.48(m, 4H), 3.59 (t, 2H), 3.72-3.81 (m, 5H, CH2), 3.97 (s, 1H), 4.59 (t,1H), 6.12, 6.26 (s, 1H), 6.75 (s, 1H), 6.85-6.96 (m, 2H), 7.15-7.51 (t,3H), 7.53-7.76 (m, 2H), 8.87 (s, 1H), 9.27 (s, 1H), 10.68 (s, 1H), 11.92(s, 1H).

Example 1 Discovery of Novel Classes of CFTR Inhibitors

A collection of 100,000 small, drug-like compounds was screened toidentify new CFTR inhibitors. As diagrammed in FIG. 1A, compounds werescreened at 25 μM in a cell-based assay of iodide influx after CFTRactivation by an agonist mixture containing forskolin, IBMX andapigenin. Initial rates of iodide influx were computed from the kineticsof fluorescence decrease following chloride replacement by iodide. Fourcompounds (FIG. 1B) reducing iodide influx by greater than 50% wereidentified, which were not related structurally to known CFTR activatorsor inhibitors. Twelve compounds reduced iodide influx by 25-50%, most ofwhich were related structurally to the compounds in FIG. 1B or to thethiazolidinones.

To select inhibitor(s) for further evaluation, dose-responsemeasurements were done for the compounds in FIG. 1B, and CFTR inhibitionwas confirmed electrophysiologically by short-circuit current analysis.K_(i) was ˜7, 5, 5 and 5 μM for compounds a-d, respectively. FIG. 1Cshows representative fluorescence and FIG. 1D shows a representation ofshort-circuit current data for compound d. 100-250 commerciallyavailable analogs of each compound class were screened to determinewhether active structural analogs exist, an important prerequisite forfollow-up compound optimization by synthesis of targeted analogs.Whereas few or no active analogs of compounds a, b and c were found,initial screening of 285 analogs of compound d (substituted glycinehydrazides, GlyH) revealed 34 analogs that inhibited CFTR-mediatediodide influx by >25% at 25 μM.

The structure-activity analysis and characterization of inhibitionmechanism, as well as the time course of action and reversibility ofaction of synthesized GlyH analogs was determined. In addition, theeffectiveness of the analogs for different CFTR activating mechanismswas also analyzed. FIG. 2A shows prompt inhibition of iodide influx inthe fluorescence and short-circuit current assays upon GlyH-101addition. Interestingly 50% of the inhibition occurred within the ˜1second addition/mixing time, with further inhibition over ˜1 minute.FIG. 2B indicates complete reversal of inhibition after GlyH-101 washoutwith >75% reversal over 5 minutes. FIG. 2C shows effective CFTRinhibition by GlyH-101 after activation by different types of agonists,including potent direct activators of CFTR that do not elevate cytosoliccAMP or inhibit phosphatase activity (CFTR_(act)-01, 08, and 10; Ma etal., J. Clin. Invest. 110:1651-1658, 2002).

Example 2 Chemistry and Structure-Activity Relationships of GlycineHydrazides

The GlyH-101 structure was modified systematically to establishstructure-activity relationships and to identify analogs with improvedCFTR inhibitory activity. FIG. 3A shows the various classes ofstructural analogues that were synthesized and tested for CFTRinhibition. Structural modifications were performed on both ends of theglycine hydrazide backbone (FIG. 3A, left, top and middle). Replacingthe glycine methylene group by a carbonyl group and replacing nitrogenby oxygen generated oxamic acid hydrazides (OxaH, right, top) and aceticacid hydrazides (AceH, right, middle), respectively. The hydrazone groupmodification produced two important series of compounds (middle, bottomand right, bottom). Also shown are compounds containing an additionalmethyl group at the hydrazone bond (top, middle), and containing a6-qunolinyl group replacing the naphthalenyl group (left, bottom).

FIG. 3B shows the reaction schemes developed for synthesis of thedifferent classes of glycine hydrazide analogs. Synthesis of GlyH-101involves reaction of 2-naphthalemine with ethyl iodoacetate followed byreactions with hydrazine hydrate and2,4-dihydroxy-3,5-dibromobenzaldehyde. A similar procedure was used formost of the remaining glycine hydrazide derivatives (listed in Table 1).The heteroaromatic analogues containing a 6-qunolinium group requireddifferent synthetic route in which 6-aminoquinoline was condensed withglyoxalic acid, and reduced using sodium cyanoborohydride (yieldingN-6-quinolineglycine, Ramamurthy et al., 1989), which was furtheresterified and reacted with hydrazine hydrate and benzaldehyde. Theoxamic acid hydrazides were synthesized starting from aromatic aminesand diethyl oxalate.

Modifications were made initially on the N-aryl (R₁) and benzaldehyde(R₂) positions (see Tables 1-4 for R- and X-group definitions and CFTRinhibition). Good CFTR inhibition was found when R₂ contained3,5-dibromo and at least one hydroxyl substituent at the 4-position(GlyH-102, 105, 114); addition of a second hydroxyl group increasedinhibition (GlyH-101, 104, 115-116). Inhibition was reduced when R₂contained 4-bromophenyl or 4-carboxyphenyl substituents (GlyH-120-121).In addition, the 4-hydroxyl group in GlyH-101 was important forinhibition since its 4-methoxy analogue GlyH-103 had little activity.Similar structure-activity results were found for GlyH-115 and GlyH-122.

R₁ group modifications were carried out, maintaining R₂ as2,4-dihydroxy-3,5-dibromophenyl and 3,5-dibromo-4-hydroxyphenyl.Analogues with R₁ as 2-naphthalenyl were much better inhibitors than R₁as 4-chlorophenyl or 4-methylphenyl. Replacement of the 2-napthalenyl ofGlyH-101 by 1-napthalenyl (GlyH-104) decreased inhibition activityten-fold, supporting the requirement of the 2-naphthalenyl substituent.GlyH-124-125, containing a 2-anthacenyl group, were less active.Replacement of 2-naphthalenyl group in GlyH-101 and GlyH-102 by morepolar heteroaromatic rings such as 6-qunolinyl gave compound with littleactivity (Gly-126-127), as did the 2-naphthoxy analogues AceH-401 andAceH-402.

X was next modified (replacing methylene), keeping 2-naphthalenyl as R₁and dibromo-dihydroxyphenyl as R₂. Introduction of a carbonyl group inGlyH-101 and GlyH-102 at X, giving OxaH-110 and OxaH-111, gave two-threefold greater inhibitory potency. FIG. 3C shows short-circuit currentanalysis of CFTR inhibition for the most active analog OxaH-110, with anapparent K_(i)˜2 μM. Replacement of CH₂ by CHCH₃ (GlyH-106-107) alsoimproved CFTR inhibition. In another structural variation, addition of amethyl group at R₃ to GlyH-102, yielding GlyH-109, gave improved CFTRinhibition. Modification of the N═C group in GlyH-101 and GlyH-102 toNH—CH₂ in GlyH-301 and GlyH-302, or to NH—CO in GlyH-201 and GlyH-202,reduced CFTR inhibitory potency.

TABLE 1 Structure-activity relationships of Group 1 hydrazide-containingcompounds Group I (I)

K_(i) % inhibition Compound R₁ X R₂ R₃ (μM) at 50 μM GlyH-1012-naphthalenyl CH₂ 3,5-di-Br-2,4-di-OH—Ph H 5 95 GlyH-102 2-naphthalenylCH₂ 3,5-di-Br-4-OH—Ph H 5 98 GlyH-103 2-naphthalenyl CH₂3,5-di-Br-2-OH-4-OMe—Ph H 20 56 GlyH-104 1-naphthalenyl CH₂3,5-di-Br-2,4-di-OH—Ph H 12 86 GlyH-105 1-naphthalenyl CH₂3,5-di-Br-4-OH—Ph H 15 87 GlyH-106 2-naphthalenyl CHCH₃3,5-di-Br-2,4-di-OH—Ph H 6 91 GlyH-107 2-naphthalenyl CHCH₃3,5-di-Br-4-OH—Ph H 10 80 GlyH-108 2-naphthalenyl CH₂3,5-di-Br-2,4-di-OH—Ph CH₃ 10 81 GlyH-109 2-naphthalenyl CH₂3,5-di-Br-4-OH—Ph CH₃ 2.5 100 OxaH-110 2-naphthalenyl CO3,5-di-Br-2,4-di-OH—Ph H 2 86 OxaH-111 2-naphthalenyl CO3,5-di-Br-4-OH—Ph H 2.5 52 OxaH-112 2-naphthalenyl CO3,5-di-Br-2,4-di-OH Ph CH₃ 3 95 OxaH-113 2-naphthalenyl CO3,5-di-Br-4-OH—Ph CH₃ 3 90 GlyH-114 4-Cl—Ph CH₂ 3,5-di-Br-4-OH—Ph H 5 95GlyH-115 4-Cl—Ph CH₂ 3,5-di-Br-2,4-di-OH Ph H 5 91 GlyH-116 4-Me—Ph CH₂3,5-di-Br-2,4-di-OH Ph H 10 79 GlyH-117 2-Me—Ph CH₂ 3,5-di-Br-2,4-di-OHPh H GlyH-118 1-naphthalenyl CH₂ 3-Br-4-OH—Ph H GlyH-119 2-naphthalenylCH₂ 2,4-di-OH—Ph H GlyH-120 2-naphthalenyl CH₂ 4-Br—Ph H GlyH-1212-naphthalenyl CH₂ 4-carboxy-Ph H GlyH-122 4-Cl—Ph CH₂3,5-di-Br-2-OH-4-OMe—Ph H GlyH-123 4-Cl—Ph CH₂ 2,4-di-OH—Ph H GlyH-1242-anthracenyl CH₂ 3,5-di-Br-2,4-di-OH Ph H GlyH-125 2-anthracenyl CH₂3,5-di-Br-4-OH—Ph H GlyH-126 6-quinolinyl CH₂ 3,5-di-Br-2,4-di-OH Ph HGlyH-127 6-quinolinyl CH₂ 3,5-di-Br-4-OH—Ph H

TABLE 2 Structure-activity relationships of Group 2 hydrazide-containingcompounds Group II (II)

K_(i) % Inhibition Compound R₁ X R₂ (μM) at 50 μM GlyH-2012-naphthalenyl CH₂ 3,5-di-Br-2,4-di-OH Ph 20 65 GlyH-202 2-naphthalenylCH₂ 3,5-di-Br-4-OH—Ph 22 57 OxaH-203 2-naphthalenyl CO3,5-di-Br-2,4-di-OH Ph >50 OxaH-204 2-naphthalenyl CO 3,5-di-Br-4-OH—Ph>50

TABLE 3 Structure-activity relationships of Group 3 hydrazide-containingcompounds Group III (III)

K_(i) % Inhibition Compound R₁ X R₂ (μM) at 50 μM GlyH-3012-naphthalenyl CH₂ 3,5-di-Br-2,4-di-OH Ph ~50 50 GlyH-302 2-naphthalenylCH₂ 3,5-di-Br-4-OH—Ph ˜50 55 OxaH-303 2-naphthalenyl CO3,5-di-Br-2,4-di-OH Ph 10 70 OxaH-304 2-naphthalenyl CO3,5-di-Br-4-OH—Ph 12 78

TABLE 4 Structure-activity relationships of Group 4 hydrazide-containingcompounds Group IV (IV)

Com- K_(i) % Inhibition pound R₁ R₂ (μM) at 50 μM AceH-401 2-naphthoxy3,5-di-Br-2,4-di-OH Ph 21 84 AceH-402 2-naphthoxy 3,5-di-Br-4-OH—Ph 1786 AceH-403 4-Me—Ph 3,5-di-Br-2,4-di-OH Ph 10 54 AceH-404 4-Me—Ph3,5-di-Br-4-OH—Ph 15 63(Tables 1-4: K_(i) indicates the concentration giving 50% inhibition ofCFTR Cl⁻ conductance by short-circuit current analysis onCFTR-expressing FRT cells.)

Example 3 Patch-Clamp Analysis of CFTR Inhibition Mechanism

The mechanism of CFTR block by GlyH-101 was studied using the whole-cellconfiguration of the patch-clamp technique. After maximal activation ofCFTR in stably transfected FRT cells by 5 μM forskolin, current-voltagerelationships were measured at GlyH-101 concentrations from 0 to 50 μM.Representative original current recordings are shown in FIG. 4A. In theabsence of inhibitor (left panel), membrane current increased linearlywith voltage and did not show relaxation phenomena, as expected for pureCFTR Cl⁻ currents. Extracellular perfusion with 10 μM GlyH-101 producedan immediate reduction in current that was strongly dependent onmembrane potential (FIG. 4A, right panel). At more positive membranepotentials outward positive currents (Cl⁻ movement into the cell) werereduced compared to inward currents. FIG. 4B shows current-voltagerelationships for GlyH-101 concentrations of 0 (control), 10 and 30 μM,and after washout of 30 μM GlyH-101 (recovery). Data for thethiazolidinone3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(referred to herein as CFTR_(inh)-172) (5 μM) is shown for comparison.The current-voltage relationship was linear in the absence of inhibitor,after GlyH-101 washout, and after inhibition by CFTR_(inh)-172, whereasGlyH-101 inhibition at submaximal concentrations produced inwardrectification. FIG. 4C summarizes percentage CFTR current block as afunction of GlyH-101 concentration at different membrane voltages.GlyH-101 inhibitory potency was reduced at more negative voltages, withapparent K_(i) of 1.4, 3.8, 5.0, and 5.6 μM for voltages of +60, +20,−20 and −60 mV, respectively (Hill coefficients, n_(H)=0.5, 0.7, 1.3,1.8).

Cell-attached patch-clamp experiments were carried out to investigatethe mechanism of GlyH-101 block of CFTR Cl⁻ current at thesingle-channel level. FIG. 4D shows a GlyH-101 concentration-dependentreduction in CFTR channel activity without a change in single channelconductance. Mean channel open time was remarkably reduced with theappearance of brief closures during the open bursts whose frequencyincreased with GlyH-101 concentration. In the absence of the inhibitor,mean channel open time was 264±11 ms (SE, n=10). Mean channel open timesat +60 mV at 0.4, 1, and 5 μM GlyH-101 were reduced to 181±29, 38±5, and13±2 ms, respectively (n=5; p<0.01 for all concentrations vs. control).

The kinetic and electrophysiological data indicate thathydrazide-containing compounds block CFTR Cl⁻ conductance by occludingthe CFTR anion pore at or near the external membrane surface. Unlike allother CFTR inhibitors, including the thiazolidinone CFTR_(inh)-172, CFTRblock by the hydrazide-containing GlyH-101 produced inwardly rectifyingCFTR Cl⁻ currents. Compared to CFTR_(inh)-172, GlyH-101 is 50-fold morewater soluble and rapidly acting/reversible when added to or removedfrom the extracellular solution, consistent with its action at theexternal-facing surface of CFTR. Structure-activity analysis of a seriesof targeted hydrazide-containing analogs defined the structuraldeterminants for CFTR inhibition and provided analogs with greater CFTRinhibitory potency, the best being OxaH-110 with Ki˜2 μM. Although themost potent thiazolidinone CFTR_(inh)-172 has Ki of 0.2-0.3 μM inpermeabilized cell preparations, its Ki is 2-5 μM in most intactepithelial cells because of the interior negative membrane potentialwhich reduces its concentration in cytoplasm. Thus, thehydrazide-containing compounds are as or more potent than thethiazolidinones, and like the thiazolidinones they block CFTR in nasaland intestinal epithelia in vivo.

Patch-clamp studies indicated that CFTR inhibition by GlyH-101 issensitive to membrane potential. At sub-maximal concentrations ofGlyH-101 there was marked inward rectification in the CFTRcurrent-voltage relationship indicating that Cl⁻ flux from theextracellular to the intracellular side of the membrane is more stronglyblocked than that in the opposite direction. The apparent Ki increasedapproximately four-fold as applied potential was varied from +60 to −60mV. Since GlyH-101 is negatively charged at pH 6-8, the simplestinterpretation of these data is that GlyH-101 inhibition involves directinteraction with the channel pore at the extracellular side of themembrane. Accordingly, negative membrane potentials reduce theinhibitory efficacy of the negatively charged GlyH-101 by electrostaticrepulsion, which drives the compound outside of the pore. In contrast,the open channel blocker glibenclamide, which is thought to act from theintracellular side of the CFTR pore (Sheppard & Robinson, 1997 J.Physiol., 503:333-346), produces outward rectification of CFTRcurrent-voltage relationship (Zhou et al., 2002, J. Gen. Physiol.,120:647-662).

Analysis of GlyH-101 dose-response data also revealed an increase inapparent Hill coefficient at more negative membrane potentials,demonstrating the possibility of more than one inhibitor binding sitewithin the pore and/or cooperative interaction between inhibitormolecules, as reported previously for other ion channels (Pottosin etal., 1999, Biophys. J., 77:1973-1979; Brock et al., 2001, J. Gen.Physiol. 118:113-134). In support of the hypothesis that GlyH-101 is anopen channel blocker, cell-attached patch-clamp experiments revealedfast closures within bursts of channel openings. The frequency of fastclosures increased with GlyH-101 concentration, producing a reduction inmean channel open time as found for glibenclamide (Sheppard & Robinson,1997J. Physiol., 503:333-346). The appearance of closure events on themillisecond time scale classifies GlyH-101 as an “intermediate”-typechannel blocker, similar to glibenclamide; in contrast, “fast” blockersreduce apparent single channel conductance, and “slow” blockers thatcause closures of many seconds duration. In whole-cell patch-clamp andshort-circuit current experiments, CFTR Cl⁻ conductance was fullyinhibited at high concentrations (≧30 μM) of GlyH-101. Together theseresults demonstrate that the GlyH-101 inhibition mechanism involvesdirect CFTR pore occlusion at a site at or near the extracellular-facingpore surface.

Example 4 Physical Properties of Glycine Hydrazides

Interpretation of the voltage-dependent inhibition mechanism requiresknowledge of the GlyH-101 ionic species that interacts with CFTR.Short-circuit studies indicated that the K_(i) for GlyH-101 inhibitionof CFTR Cl⁻ current was independent of pH in the range 6-8 (not shown),where the compound is highly water soluble (0.8-1.3 mM in water, 22°C.). The possible titrable groups on GlyH-101 in the pH range 3-10include the secondary glycinyl amine and the resorcinolic hydroxyls.Spectrophotometric titration of GlyH-101 indicated at least twoprotonation/deprotonations at pH between 4 and 9 (FIG. 5A, top panel).To assign pKa values, GlyH-101 analogs that lacked one or more titrablegroups were synthesized. Removal of the secondary amine (AceH-403) hadlittle effect on the titration, with only a minor left-shift of theascending portion of the curve, suggesting a pKa of ˜5.5 for titrationof the first phenolic hydroxyl. Removal of one ortho hydroxyl (GlyH-102)eliminated the descending portion of the curve, confirming the pKa of˜5.5 for the first para hydroxyl and ˜8.5 for the second ortho hydroxyl.Removal of the aromatic ring containing the resorcinolic hydroxyls(ethyl N-(2-napthalenyl) glycinate, FIG. 5A, bottom panel) indicated apKa ˜4.7 for the residual secondary amine. From these data the deducedequilibria among the ionic forms of GlyH-101 is shown in FIG. 5B.GlyH-101 exists primarily as a singly charged anion at pH between 6 and8.

Example 5 CFTR Inhibition in Mice In Vivo

Inhibition of CFTR-dependent airway epithelial Cl⁻ current in vivo wasdemonstrated by nasal potential difference (PD) measurements in mice.Nasal PDs were measured continuously in response to serial solutionexchanges in which amiloride was added (to block ENaC Na⁺ channels)followed by Cl⁻ replacement by gluconate (to induce Cl⁻ dependenthyperpolarization), forskolin addition (to activate CFTR) and GlyH-101addition (to inhibit CFTR). The representative PD recording in FIG. 6A(left panel) shows hyperpolarizations (more negative PDs) following lowCl⁻ and forskolin solutions, representing CFTR-independent and dependentCl⁻ currents, respectively. Topical application of GlyH-101 in theperfusate rapidly reversed the forskolin-induced hyperpolarization.Averaged results from a series of measurements are summarized in FIG. 6A(right panel). Paired analysis of PD changes (ΔPD, FIG. 6B) indicated ˜4mV hyperpolarization after forskolin with depolarization of similarmagnitude after GlyH-101; for comparison data are shown forCFTR_(inh)-172 from a previous study. In a separate series ofexperiments, nasal PDs were measured as in A except that all solutionscontained DIDS or GlyH-101. FIG. 6C shows partial inhibition by DIDS ofthe (CFTR-independent) hyperpolarization produced by low Cl⁻ (leftpanel), and substantial inhibition by GlyH-101 of the forskolin-inducedhyperpolarization (right panel). Together these results indicate rapidinhibition of upper airway CFTR Cl⁻ conductance by topical GlyH-101.

The efficacy of GlyH-101 in inhibiting cAMP/cholera toxin-inducedintestinal fluid secretion was also evaluated. Short-circuit currentexperiments were done in different cell types and in intact mouse ileumunder non-permeabilized conditions and in the absence of a Cl⁻ gradient.In each case CFTR was activated by CPT-cAMP after ENaC inhibition byamiloride. FIG. 7A shows similar K_(i)˜5 μM for inhibition ofcAMP-stimulated short-circuit current by GlyH-101 in T84 cells (toppanel), primary human bronchial cell cultures (middle panel), and intactmouse ileum (bottom panel). Inhibition was ˜100% at higher GlyH-101concentrations. Cholera toxin-induced intestinal fluid secretion wasmeasured in an in vivo closed-loop model in which loops for each mousewere injected with saline (control), cholera toxin (1 μg), or choleratoxin (1 μg)+GlyH-101 (0.25 μg). GlyH-101 was added to the lumen (ratherthan systemically) based on initial studies showing poor intestinalabsorption and little effect of systemically administered compound.Compared to the saline control, the cholera toxin-induced increase influid secretion over 4 hours, quantified from loop weight-to-lengthratio, was 80% reduced by GlyH-101.

Example 6 Synthesis of Highly Water Soluble CFTR Pore-Blocking Compounds

The strategy for design of highly water-soluble CFTR inhibitor compoundswith minimal intestinal absorption was to modify the structure ofGlyH-101 by addition of polar, bulky groups as shown in FIG. 8. Fromanalysis of structure-activity relationship of glycine hydrazidescompounds it was found that the minor modifications at the glycyl methylposition did not affect CFTR inhibition activity. Efficient synthesis ofhighly water soluble CFTR inhibitors were devised by utilizing adiethylbromomalonate intermediate (FIGS. 9-11). Reaction of2-naphthalenamine with diethylbromomalonate followed by subsequentreaction with hydrazine generated a versatile malonic acid dihydrazideintermediate (FIG. 9). Condensation of this dihydrazide with3,5-dibromo-2,4-dihydroxybenzaldehyde produced a key intermediatecompound 4 which on further condensation with same aldehyde produced thecompound MalH-1. Similarly, 2,4-disodium-disulfobenzaldehyde and4-sodium-sulfophenylisothiocyanate were condensed with compound 4 togenerate the compounds MalH-2 and MalH-3, respectively.

MalH-1 is structurally similar to GlyH-101 except for an additionalbenzaldehyde moiety that makes it doubly charged, bulkier and morehydrophilic. MalH-1 is water soluble to >5 mM. MalH-2 carries twodisulfonic acid groups, and MalH-3 contains one sulfonic acid moietywith hydrophilic thiourea linker. Both compounds are freely soluble(>50% wt/volume, 20° C.) in water and saline.

Intermediate compound 4 was also used to generate MalH-(PEG)_(n) andMalH-(PEG)_(n)B by condensation with various phenylisothiocyantes 6a and6b carrying PEG (FIG. 10). The intermediate compounds 6a and 6b weresynthesized by reaction of 1,4-phenylenediisothiocyante 5a andbis[(4-isothiocyanato)phenyl]methane 6b with appropriate amino-PEGs. ThePEG moiety increased water solubility to 10 mM. Another approach forsynthesis of PEG-ylated compounds involved incorporation of hydroxyethylmoiety onto glycyl methyl and further manipulating hydroxyl group tolink PEG chain (FIG. 11). Reaction of bromobuterolactone with2-naphthalenamine and subsequent reaction with hydrazine producedhydrazide 7. Using standard protection-deprotection Boc chemistry, thishydrazide was PEG-ylated by utilizing its hydroxyl group. The PEG-ylatedhydrazide 11 was condensed with aromatic aldehyde to produceGlyH-(PEG)_(n), which have similar was solubility as MalH-(PEG)_(n).

Example 7 CFTR Inhibition with Highly Water Soluble CFTR Pore-BlockingCompounds

CFTR inhibition by MalH compounds was assayed by short-circuit currentanalysis using FRT cells expressing human wildtype CFTR. Apical membranechloride current was measured after permeabilization of the cellbasolateral membrane in the presence of a transepithelial chloridegradient. As shown in FIG. 12, CFTR was activated by the cell permeantcAMP agonist CPT-cAMP and then increasing MalH compound concentrationswere added. The results show that inhibition was rapid and nearlycomplete at high MalH concentrations. In addition, the results also showthat inhibitory potencies (K_(I)) were in the range 2-8 μM.

Short-circuit current analysis in CFTR-expressing epithelial cellmonolayers showed prompt inhibition of chloride current in response tocompound addition to the luminal solution. Importantly, near 100% blockof chloride current was achieved at high inhibition concentrations.Also, the inhibitors were chemically stable in the presence ofintestinal contents, and no toxicity was seen when the inhibitors werepresent at high concentration in cell cultures or when administeredsystemically to mice. The effective CFTR block of these water solubleimpermeant compounds when added externally provides direct evidence thatthe site of the block is at the external-facing surface of CFTR.

Example 8 Intestinal Absorption and Antidiarrheal Efficacy Studies withHighly Water Soluble CFTR Pore-Blocking Compounds

Intestinal absorption was measured in mice in vivo from thedisappearance of MalH compounds from the lumens of closed mid-jejunalloops over 2 hours. In these experiments mannitol was included in theMalH-containing solutions to prevent fluid absorption. Absorption rateswere referenced against a large FITC-dextran, which was assumed toundergo no absorption over the 2 hour study. The summarized data in FIG.13, panel A, shows under 5% absorption of the MalH compounds in 2 hours,whereas the >90% of the thiazolidinone CFTR_(inh)-172 was absorbed overthis time.

Antidiarrheal efficacy was assayed in closed mid-jejunal loops in mice.Loops were injected with saline or solutions of cholera toxin containingdifferent concentrations of MalH compounds. Intestinal fluid secretionwas determined at 6 hours by measurements of loop length and weight. Thedata summary in FIG. 13, panel B, shows a loop weight-to-length ratio(corresponding to 100% inhibition) of 0.09 in saline-injected loops, and0.28 (corresponding to 0% inhibition) in cholera toxin-injected loops.The results show that each of the MalH compounds inhibited loopsecretion in a dose-dependent manner with essentially completeinhibition at the higher concentrations.

The results show that the glycine hydrazide-based CFTR inhibitorsundergo little intestinal absorption and are effective in preventingcholera toxin-induced fluid secretion in a rodent model of choleratoxin-induced fluid secretion. The advantages of antidiarrheal therapyusing a non-absorbable compound are that high concentrations can beachieved in the gut with minimal concerns about toxicity and off-targeteffects related to cellular uptake and systems absorption.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of treating a subject having a condition associated withaberrant ion transport by cystic fibrosis transmembrane conductanceregulator (CFTR) in a subject, the method comprising: administering tothe subject an efficacious amount of a hydrazide-containing compound;wherein CFTR ion transport is inhibited and the condition is treated. 2.The method of claim 1, wherein the aberrantly increased CFTR iontransport is associated with polycystic kidney disease.
 3. The method ofclaim 1, wherein the aberrantly increased CFTR ion transport isassociated with diarrhea.
 4. The method of claim 3, wherein the diarrheais secretory diarrhea.
 5. The method of claim 1, wherein thehydrazide-containing compound has the following formula (I):

wherein X is independently chosen from an alkyl group or a carbonylgroup; Y is independently chosen from a hydrogen, an alkyl group, anamide bond linker, or an ether linker; R₁ is independently chosen from asubstituted or unsubstituted phenyl group, a substituted orunsubstituted heteroaromatic group such as a quinolinyl group, asubstituted or unsubstituted anthracenyl group, and a substituted orunsubstituted naphthalenyl group; R₂ is a substituted or unsubstitutedphenyl group; and R₃ is independently chosen from hydrogen or an alkylgroup; or a pharmaceutically acceptable derivative thereof, as anindividual stereoisomer or a mixture thereof, as an individualstereoisomer or a mixture thereof, or a pharmaceutically acceptable saltthereof.
 6. The method of claim 1, wherein the hydrazide-containingcompound has the following formula (Ia):

wherein X₁ is independently chosen from a hydrogen or a substituted orunsubstituted alkyl group; Y is independently chosen from a hydrogen, analkyl group, an amide bond linker, or an ether linker; R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenor an alkyl group; wherein when X₁ is hydrogen, R₁ is a substituted orunsubstituted anthracenyl group, a substituted or unsubstituted phenylgroup, or a heteroaromatic group.
 7. The method of claim 1, wherein thehydrazide-containing compound has the following formula (Ic):

wherein Y is independently chosen from an alkyl group, an amide bondlinker, or an ether linker R₁ is independently chosen from a substitutedor unsubstituted phenyl group, a substituted or unsubstitutedheteroaromatic group such as a quinolinyl group, a substituted orunsubstituted anthracenyl group, and a substituted or unsubstitutednaphthalenyl group; R₂ is a substituted or unsubstituted phenyl group;and R₃ is independently chosen from hydrogen or an alkyl group.
 8. Themethod of claim 1, wherein the hydrazide-containing compound of formula(I) is chosen from:N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide;N-2-napthalenyl-[(3,5-dibromo-2,4,6-trihydroxyphenyl)methylene]glycinehydrazideN-(substituted-2-(napthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide,N-2-napthalenyl-[(3,5-dibromo-4-hydroxyphenyl)methylene]glycinehydrazide;N-2-napthalenyl-[(3,5-dibromo-2-hydroxy-4-mthoxyphenyl)methylene]glycinehydrazide;N-1-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide;N-1-napthalenyl-[(3,5-dibromo-2,4,6-trihydroxyphenyl)methylene]glycinehydrazide;N-(substituted-1-napthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide;N-1-napthalenyl-[(3,5-dibromo-4-hydroxyphenyl)methylene]glycinehydrazide;N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propionicacid hydrazide;N-2-napthalenyl-[(3,5-dibromo-4-hydroxyphenyl)methylene]propionic acidhydrazide;N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)ethylene]glycinehydrazide;N-2-napthalenyl-[(3,5-dibromo-4-hydroxyphenyl)ethylene]glycinehydrazide;N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]oxamic acidhydrazide;N-2-napthalenyl-[(3,5-dibromo-4-hydroxyphenyl)methylene]oxamic acidhydrazide;N-2-napthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)ethylene]oxamic acidhydrazide; N-2-napthalenyl-[(3,5-dibromo-4-hydroxyphenyl)ethylene]oxamicacid hydrazide;4-chlorophenyl-[(3,5-dibromo-4-hydroxyphenyl)methylene]glycinehydrazide;4-chlorophenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide;4-methylphenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide;2-methylphenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide; N-1-napthalenyl-[(3-bromo-4-hydroxyphenyl)methylene]glycinehydrazide; N-2-napthalenyl-[(2,4-dihydroxyphenyl)methylene]glycinehydrazide; N-2-napthalenyl-[(4-bromophenyl)methylene]glycine hydrazide;N-2-napthalenyl-[(4-carboxy-phenyl)methylene]glycine hydrazide;4-chlorophenyl-[(3,5-dibromo-2-hdroxy-4-methoxyphenyl)methylene]glycinehydrazide; 4-chlorophenyl-[(2,4-dihydroxyphenyl)methylene]glycinehydrazide;N-2-anthracenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide;N-2-anthracenyl-[(3,5-dibromo-4-hydroxyphenyl)methylene]glycinehydrazide;N-6-quinolinyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycinehydrazide;N-6-quinolinyl-[(3,5-dibromo-4-hydroxyphenyl)methylene]glycinehydrazide,N-(heteroaryl)-[(3,5-dibromo-4-hydroxyphenyl)methylene]glycinehydrazide;2-naphthalenylamino-bis[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]propanedioicacid dihydrazide;2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][(2,4-disodium-disulfophenyl)methylene]propanedioicacid dihydrazide;2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-(4-sodium-sulfophenyl)-thioureido]propanedioicacid dihydrazide;2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-[4-(3-(PEG)_(n)-thioureido)phenyl)-thioureido]propanedioicacid dihydrazide; [2-(2-naphthalenylamino)-4-(PEG-amino)]butyric acidhydrazide; or2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene][3-[4-((3-(PEG)_(n)-thioureido)phenyl-methyl)phenyl)-thioureido]propanedioicacid dihydrazide [MalH-(PEG)_(n) B].
 9. A method for inhibiting theactivity of cystic fibrosis transmembrane conductance regulator (CFTR)protein in a cell in an in vitro assay, comprising contacting the cellwith a hydrazide-containing compound in an amount effective to inhibitCFTR activity, wherein the hydrazide-containing compound has thefollowing formula (I):

wherein X is independently chosen from an alkyl group or a carbonylgroup; Y is independently chosen from a hydrogen, an alkyl group, anamide bond linker, or an ether linker; R₁ is independently chosen from asubstituted or unsubstituted phenyl group, a substituted orunsubstituted heteroaromatic group such as a quinolinyl group, asubstituted or unsubstituted anthracenyl group, and a substituted orunsubstituted naphthalenyl group; R₂ is a substituted or unsubstitutedphenyl group; and R₃ is independently chosen from hydrogen or an alkylgroup; or a pharmaceutically acceptable derivative thereof, as anindividual stereoisomer or a mixture thereof, as an individualstereoisomer or a mixture thereof; or a pharmaceutically acceptable saltthereof.
 10. A method for inhibiting the activity of cystic fibrosistransmembrane conductance regulator (CFTR) protein in a cell in an invitro assay, comprising contacting the cell with a hydrazide-containingcompound in an amount effective to inhibit CFTR activity, wherein thehydrazide-containing compound has the following formula (Ia):

wherein X₁ is independently chosen from a hydrogen or a substituted orunsubstituted alkyl group; Y is independently chosen from a hydrogen, analkyl group, an amide bond linker, or an ether linker; R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenor an alkyl group; wherein when X₁ is hydrogen, R₁ is a substituted orunsubstituted anthracenyl group, a substituted or unsubstituted phenylgroup, or a heteroaromatic group.
 11. A method for inhibiting theactivity of cystic fibrosis transmembrane conductance regulator (CFTR)protein in a cell in an in vitro assay, comprising contacting the cellwith a hydrazide-containing compound in an amount effective to inhibitCFTR activity, wherein the hydrazide-containing compound has thefollowing formula (Ic):

wherein Y is independently chosen from an alkyl group, an amide bondlinker, or an ether linker R₁ is independently chosen from a substitutedor unsubstituted phenyl group, a substituted or unsubstitutedheteroaromatic group such as a quinolinyl group, a substituted orunsubstituted anthracenyl group, and a substituted or unsubstitutednaphthalenyl group; R₂ is a substituted or unsubstituted phenyl group;and R₃ is independently chosen from hydrogen or an alkyl group.
 12. Amethod for producing a cystic fibrosis (CF) phenotype in a non-humananimal, wherein the method comprises administering to the non-humananimal a hydrazide-containing compound in an amount effective to inhibitCFTR ion transport, wherein the hydrazide-containing compound has thefollowing formula (I):

wherein X is independently chosen from an alkyl group or a carbonylgroup; Y is independently chosen from a hydrogen, an alkyl group, anamide bond linker, or an ether linker; R₁ is independently chosen from asubstituted or unsubstituted phenyl group, a substituted orunsubstituted heteroaromatic group such as a quinolinyl group, asubstituted or unsubstituted anthracenyl group, and a substituted orunsubstituted naphthalenyl group; R₂ is a substituted or unsubstitutedphenyl group; and R₃ is independently chosen from hydrogen or an alkylgroup; or a pharmaceutically acceptable derivative thereof, as anindividual stereoisomer or a mixture thereof, as an individualstereoisomer or a mixture thereof; or a pharmaceutically acceptable saltthereof.
 13. A non-human animal having a cystic fibrosis transmembraneconductance regulator (CFTR) deficiency produced by the method of claim12, wherein the deficiency is produced by administration of thehydrazide-containing compound to the animal.
 14. A method for producinga cystic fibrosis (CF) phenotype in a non-human animal, wherein themethod comprises administering to the non-human animal ahydrazide-containing compound in an amount effective to inhibit CFTR iontransport, wherein the hydrazide-containing compound has the followingformula (Ia):

wherein X₁ is independently chosen from a hydrogen or a substituted orunsubstituted alkyl group; Y is independently chosen from a hydrogen, analkyl group, an amide bond linker, or an ether linker; R₁ isindependently chosen from a substituted or unsubstituted phenyl group, asubstituted or unsubstituted heteroaromatic group such as a quinolinylgroup, a substituted or unsubstituted anthracenyl group, and asubstituted or unsubstituted naphthalenyl group; R₂ is a substituted orunsubstituted phenyl group; and R₃ is independently chosen from hydrogenor an alkyl group; wherein when X₁ is hydrogen, R₁ is a substituted orunsubstituted anthracenyl group, a substituted or unsubstituted phenylgroup, or a heteroaromatic group.
 15. A non-human animal having a cysticfibrosis transmembrane conductance regulator (CFTR) deficiency producedby the method of claim 14, wherein the deficiency is produced byadministration of the hydrazide-containing compound to the animal.
 16. Amethod for producing a cystic fibrosis (CF) phenotype in a non-humananimal, wherein the method comprises administering to the non-humananimal a hydrazide-containing compound in an amount effective to inhibitCFTR ion transport, wherein the hydrazide-containing compound has thefollowing formula (Ic):

wherein Y is independently chosen from an alkyl group, an amide bondlinker, or an ether linker R₁ is independently chosen from a substitutedor unsubstituted phenyl group, a substituted or unsubstitutedheteroaromatic group such as a quinolinyl group, a substituted orunsubstituted anthracenyl group, and a substituted or unsubstitutednaphthalenyl group; R₂ is a substituted or unsubstituted phenyl group;and R₃ is independently chosen from hydrogen or an alkyl group.
 17. Anon-human animal having a cystic fibrosis transmembrane conductanceregulator (CFTR) deficiency produced by the method of claim 16, whereinthe deficiency is produced by administration of the hydrazide-containingcompound to the animal.